Ketogulonigenium endogenous plasmids

ABSTRACT

The present invention relates, in general, to a novel genus of bacteria known as Ketogulonigenium. The present invention further relates to transformed Ketogulonigenium, and methods of transforming Ketogulonigenium. The present invention also relates to nucleic acid molecules, and vectors.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This non-provisional application is related to provisionalapplication No. 60/194,627, filed Apr. 5, 2000, the content of which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates, in general, to a novel genus ofbacteria known as Ketogulonigenium. The present invention furtherrelates to transformed Ketogulonigenium, and methods of transformingKetogulonigenium. The present invention also relates to nucleic acidmolecules, and vectors.

[0004] 2. Background Art

[0005] The exploitation of microorganisms to synthesize vitamin C or itschemical pathway intermediates has both economic and ecologicaladvantages. One key intermediate in vitamin C synthesis is2-keto-L-gulonic acid (2-KLG), which is easily converted chemically toL-ascorbic acid (vitamin C) by esterification followed by lactonization(Delic, V. et al., “Microbial reactions for the synthesis of vitamin C(L-ascorbic acid,” in Biotechnology of Vitamins, Pigments and GrowthFactors, Vandamme, E. J., ed., Elsevier Applied Science (London & NewYork) pp. 299-336 (1989)). Members of a number of bacterial genera havebeen identified that produce 2-KLG from the oxidation of L-sorbose orsorbitol. Such 2-KLG producing genera include the acidogenic,alpha-proteobacteria Gluconobacter and Acetobacter, thegamma-proteobacteria Pseudomonas, Escherichia, Klebsiella, Serratia andXanthmonas and the Gram positive Bacillus, Micrococcus andPseudogluconobacter (Imai, K. et al., U.S. Pat. No. 4,933,289 (1990),Sugisawa, H. et al., “Microbial production of 2-keto-L-gulonic acid fromL-sorbose and D-sorbitol by Gluconobacter melanogenus,” Agric. Biol.Chem. 54:1201-1209 (1990), Yin, G. et al., U.S. Pat. No. 4,935,359(1990), Shirafuji, et al., U.S. Pat. No. 4,876,195 (1989) and Nogami, I.et al., U.S. Pat. No. 5,474,924 (1995)).

[0006] To aid in increasing the yield of bacterial products, attemptshave been made to exploit endogenous plasmids within microorganismstrains. (Beppu, T. et al., U.S. Pat. No. 5,580,782 (1996), Fujiwara, A.et al., U.S. Pat. No. 5,399,496 (1995); Tonouchi et al, U.S. Pat. No.6,127,174 (2000), Hoshino, T. et at., U.S. Pat. No. 6,127,156 (2000)).

BRIEF SUMMARY OF THE INVENTION

[0007] One aspect of the present invention provides an isolated orpurified nucleic acid molecule comprising a polynucleotide having anucleotide sequence at least 95% identical to a nucleotide sequence of aKetogulonigenium plasmid replicon found on the endogenous plasmidcontained in NRRL Deposit No. B-21627 and at least one exogenousnucleotide sequence.

[0008] Further embodiments of the invention include an isolated orpurified nucleic acid molecule comprising a polynucleotide having anucleotide sequence at least 90% identical, and more preferably at least95%, 97%, 98% or 99% identical, to any of the above nucleotidesequences, or a polynucleotide which hybridizes under stringenthybridization conditions to a polynucleotide having a nucleotidesequence as in the above. The polynucleotide which hybridizes does nothybridize under stringent hybridization conditions to a polynucleotidehaving a nucleotide sequence consisting of only A residues or of only Tresidues.

[0009] The present invention relates, in general, to a novel genus ofbacteria known as Ketogulonigenium. The present invention furtherrelates to Ketogulonigenium comprising a transgene (recombinant DNA),comprising an endogenous plasmid. The invention also relates to a methodfor transforming Ketogulonigenium comprising conjugative transfer of avector from E. coli to Ketogulonigenium, and to a method fortransforming Ketogulonigenium comprising electroporation.

[0010] The invention provides a nucleic acid molecule comprising anucleotide sequence at least 95% identical to a Ketogulonigeniumendogenous plasmid contained in NRRL Deposit No. B-21627. The inventionalso provides a nucleic acid molecule comprising a polynucleotide havinga sequence at least 95% identical to a Ketogulonigenium replicon foundon an endogenous plasmid contained in NRRL Deposit No. B-21627. Theinvention further provides a vector comprising a nucleic acid moleculecomprising a nucleotide sequence of a Ketogulonigenium replicon found onan endogenous plasmid contained in NRRL Deposit No. B-21627.

[0011] Further advantages of the present invention will be clear fromthe description that follows.

BRIEF DESCRIPTION OF THE FIGURES

[0012] FIGS. 1A-1E show the nucleotide (SEQ ID NO:1) sequence of anendogenous plasmid, determined by sequencing of the endogenous plasmid(pADMX6L1), contained in NRRL Deposit No. B-21627. The nucleotide has asequence of about 7029 nucleic acid residues.

[0013] FIGS. 2A-2C show the nucleotide (SEQ ID NO:2) sequence of anendogenous plasmid, determined by sequencing of the endogenous plasmid(pADMX6L2), contained in NRRL Deposit No. B-21627. The nucleotide has asequence of about 4005 nucleic acid residues.

[0014] FIGS. 3A-3N show the nucleotide (SEQ ID NO:3) sequence of anendogenous plasmid, determined by sequencing of the endogenous plasmid(pADMX6L3), contained in NRRL Deposit No. B-21627. The nucleotide has asequence of about 19,695 nucleic acid residues.

[0015] FIGS. 4A-4C show the nucleotide (SEQ ID NO:4) sequence of anendogenous plasmid, determined by sequencing of the endogenous plasmid(pADMX6L4), contained in NRRL Deposit No. B-21627. The nucleotide has asequence of about 4211 nucleic acid residues.

[0016] FIGS. 5A-5B show the nucleotide (SEQ ID NO:5) sequence of thereplicon on an endogenous plasmid (pADMX6L1), determined by homology ofamino acid sequences encoded by the endogenous plasmid to knownreplication proteins, contained in NRRL Deposit No. B-21627. Thenucleotide has a sequence of about 1456 nucleic acid residues.

[0017] FIGS. 6A-6B show the nucleotide (SEQ ID NO:6) sequence of the thereplicon on an endogenous plasmid (pADMX6L2), determined by homology ofamino acid sequences encoded by the endogenous plasmid to knownreplication proteins, contained in NRRL Deposit No. B-21627. Thenucleotide has a sequence of about 2401 nucleic acid residues.

[0018] FIGS. 7A-7B show the nucleotide (SEQ ID NO:7) sequence of the thereplicon on an endogenous plasmid (pADMX6L3), determined by homology ofamino acid sequences encoded by the endogenous plasmid to knownreplication proteins, contained in NRRL Deposit No. B-21627. Thenucleotide has a sequence of about 2029 nucleic acid residues.

[0019]FIG. 8 shows the amino acid sequence (SEQ ID NO:8) of areplication protein encoded by an endogenous plasmid (pADMX6L1)determined from the nucleotide sequence of pADMX6L1 contained in NRRLB-21627. The polypeptide has a sequence of about 151 amino acids inlength.

[0020]FIG. 9 shows the amino acid sequence (SEQ ID NO:9) of areplication protein encoded by an endogenous plasmid (pADMX6L2)determined from the nucleotide sequence of pADMX6L2 contained in NRRLB-21627. The polypeptide has a sequence of about 466 amino acids inlength.

[0021]FIG. 10 shows the amino acid sequence (SEQ ID NO:10) of areplication protein encoded by an endogenous plasmid (pADMX6L3)determined from the nucleotide sequence of pADMX6L3 contained in NRRLB-21627. The polypeptide has a sequence of about 342 amino acids inlength.

DETAILED DESCRIPTION OF THE INVENTION

[0022] Unless otherwise indicated, all nucleotide sequences determinedby sequencing a DNA molecule herein were determined using an automatedDNA sequencer (such as the ABI Prism 3700). Therefore, as is known inthe art for any DNA sequence determined by this automated approach, anynucleotide sequence determined herein may contain some errors.Nucleotide sequences determined by automation are typically at leastabout 90% identical, more typically at least about 95% to at least about99.9% identical to the actual nucleotide sequence of the sequenced DNAmolecule.

[0023] Unless otherwise indicated, each “nucleotide sequence” set forthherein is presented as a sequence of deoxyribonucleotides (abbreviatedA, G, C and T). However, by “nucleotide sequence” of a nucleic acidmolecule or polynucleotide is intended, for a DNA molecule orpolynucleotide, a sequence of deoxyribonucleotides, and for an RNAmolecule or polynucleotide, the corresponding sequence ofribonucleotides (A, G, C and U) where each thymidine deoxynucleotide (T)in the specified deoxynucleotide sequence in is replaced by theribonucleotide uridine (U). For instance, reference to an RNA moleculehaving the sequence of SEQ ID NO:1 set forth using deoxyribonucleotideabbreviations is intended to indicate an RNA molecule having a sequencein which each deoxynucleotide A, G or C of SEQ ID NO:1 has been replacedby the corresponding ribonucleotide A, G or C, and each deoxynucleotideT has been replaced by a ribonucleotide U.

[0024] As indicated, nucleic acid molecules of the present invention maybe in the form of RNA, such as mRNA, or in the form of DNA, including,for instance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

[0025] By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in avector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically.

[0026] In another aspect, the invention provides an isolated nucleicacid molecule comprising a polynucleotide which hybridizes understringent hybridization conditions to a portion of the polynucleotide ina nucleic acid molecule of the invention described above, for instance,in the endogenous plasmids contained in NRRL Deposit No. B-21627. By“stringent hybridization conditions” is intended overnight incubation at42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1×SSC at about 65° C. Bya polynucleotide which hybridizes to a “portion” of a polynucleotide isintended a polynucleotide (either DNA or RNA) hybridizing to at leastabout 15 nucleotides (nt), and more preferably at least about 20 nt,still more preferably at least about 30 nt, and even more preferablyabout 30-70 nt of the reference polynucleotide. These are useful asdiagnostic probes and primers.

[0027] Of course, polynucleotides hybridizing to a larger portion of thereference polynucleotide (e.g., the deposited endogenous plasmid), forinstance, a portion 50-750 nt in length, or even to the entire length ofthe reference polynucleotide, also useful as probes according to thepresent invention, as are polynucleotides corresponding to most, if notall, of the nucleotide sequence of the deposited DNA or the nucleotidesequence as shown in FIG. 1 (SEQ ID NO:1). By a portion of apolynucleotide of “at least 20 nt in length,” for example, is intended20 or more contiguous nucleotides from the nucleotide sequence of thereference polynucleotide, (e.g., the deposited DNA or the nucleotidesequence as shown in FIG. 1 (SEQ ID NO:1)). As indicated, such portionsare useful diagnostically either as a probe according to conventionalDNA hybridization techniques or as primers for amplification of a targetsequence by the polymerase chain reaction (PCR), as described, forinstance, in Molecular Cloning, A Laboratory Manual, 2nd. edition,edited by Sambrook, J., Fritsch, E. F. and Maniatis, T., (1989), ColdSpring Harbor Laboratory Press, the entire disclosure of which is herebyincorporated herein by reference.

[0028] By a polynucleotide having a nucleotide sequence at least, forexample, 95% “identical” to a reference nucleotide sequence is intendedthat the nucleotide sequence of the polynucleotide is identical to thereference sequence except that the polynucleotide sequence may includeup to five point mutations per each 100 nucleotides of the referencenucleotide sequence. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence.

[0029] As a practical matter, whether any particular nucleic acidmolecule is at least 90%, 95%, 97%, 98% or 99% identical to, forinstance, the nucleotide sequence shown in FIG. 1 or to the nucleotidesequence of the deposited endogenous plasmid can be determinedconventionally using known computer programs such as the FastA program.FastA does a Pearson and Lipman search for similarity between a querysequence and a group of sequences of the same type nucleic acid.Professor William Pearson of the University of Virginia Department ofBiochemistry wrote the FASTA program family (FastA, TFastA, FastX,TFastX and SSearch). In collaboration with Dr. Pearson, the programswere modified and documented for distribution with GCG Version 6.1 byMary Schultz and Irv Edelman, and for Versions 8 through 10 by SueOlson.

[0030] The present invention provides Ketogulonigenium, comprising atransgene (recombinant DNA) comprising an endogenous Ketogulonigeniumplasmid. Preferably, the endogenous Ketogulonigenium plasmid iscontained in Deposit No. NRRL B-21627. As used herein, a transgene isdefined as a transplanted nucleotide sequence which is exogenous, ornon-native, to the host. An exogenous nucleotide sequence, as used inthe current context, is a nucleotide sequence which is not found inDeposit No. NRRL B-21627. Thus, the term exogenous nucleotide sequenceis meant to encompass a nucleotide sequence that is foreign to DepositNo. NRRL B-21627, as well as a nucleotide sequence endogenous, ornative, to Deposit No. NRRL B-21627 that has been modified. Modificationof the endogenous nucleotide sequence may include, for instance,mutation of the native nucleotide sequence or any of its regulatoryelements. As used herein, mutation is defined as any change in thewild-type sequence of genomic or plasmid DNA. An additional form ofmodification may also include fusion of the endogenous nucleotidesequence to a nucleotide sequence that is normally not present, inrelation to the endogenous nucleotide sequence. The transgene may beregulated by its normal promoter, or more commonly, by a promoter thatnormally regulates a different gene. The invention also provides amethod for producing transformed Ketogulonigenium, comprisingtransforming Ketogulonigenium with a transgene, comprising, part or allof an endogenous Ketogulonigenium plasmid. Preferably, the endogenousKetogulonigenium plasmid is contained in Deposit No. NRRL B-21627.

[0031] The term replicon as used herein is meant to encompass a DNAsequence comprising those genes and gene expression control elementssuch as promoters and terminators, other DNA sequence features such asshort sequence repeats (iterons), origins of plasmid replication (ori ororiV sites), or other DNA sequence features that are required to supportthe autonomous replication of a circular DNA molecule in a bacterialhost (Chapter 122, pp. 2295-2324, in Escherichia coli and Salmonella:Cellular and Molecular Biology, 2^(nd) Edition, Frederick C. Neidhardt,Ed., ASM Press (1996)). The requirements of a replicon can vary from aslittle as a short ori sequence in the case of plasmids that do notrequire their own replication proteins, to larger sequences containingone or more plasmid-borne replication genes.

[0032] The definition of a transformed cell, as used herein, is a cellwhere DNA has been inserted into a bacterial cell. The transformation ofKetogulonigenium may be transient or stable. Preferably, the inventionprovides a method for producing stably transformed Ketogulonigenium. Asused herein, a stably transformed cell is a cell wherein a transgene istransmitted to every successive generation. A preferred embodiment ofthe present invention is that Ketogulonigenium is transformed viaelectroporation. An additional preferred embodiment of the presentinvention is that Ketogulonigenium is transformed by the process ofconjugation, including, for instance, bi-parental and tri-parentalconjugation. Conjugation, as used herein, is the process by whichbacteria transfer DNA from a donor cell to a recipient cell throughcell-to cell contact.

[0033] The present invention relates to a novel genus of bacteria,comprising the ADMX6L strain, designated as Ketogulonigenium. Thepresent inventors have discovered novel strains of bacteria, notbelonging to any known genera, that produce 2-keto-L-gulonic acid fromsorbitol.

[0034] Ketogulonigenium (Ke.to.gu.lo.ni.gen'.i.um. M.L. n. acidumketogulonicum ketogulonic acid; Gr. V. gennaio to produce; M.L. n.ketogulonigenium ketogulonic acid producing) is gram negative,facultatively anaerobic, motile or non-motile, has ovoid to rod-shapedcells, 0.8-1.3 μm long, 0.5-0.7 μm in diameter, with tapered ends,occurring as single cells, pairs and occasionally short chains. Somestrains form elongated cells (up to 30 μm in length) on TSB. Flagellaand fimbrae have been observed. Colonies are tan colored, smooth,circular, entire, raised to convex, 1-2 mm in diameter with a diffusablebrown pigment after 48 hrs incubation. Oxidase and catalase reactionsare positive. Optimum temperature range is 27 to 31° C., optimum pHrange is 7.2 to 8.5 and optimum Na⁺ concentration is 117-459 mM.Chemoorganotrophic. Carbon sources utilized include arabinose,cellobiose, fructose, glucose, glycerol, inositol, lactose, maltose,mannitol, mannose, rhamnose, sorbitol, sorbose, sucrose, trehalose,pyruvate and succinate. Favored carbon sources are inositol, mannitol,glycerol, sorbitol, lactose and arabinose. All strains examined produce2-keto-L-gulonic acid from L-sorbose. Major cellular fatty acids are16:0 and 18:1 ω7c/ω9t/ω12t and the mol % DNA G+C is 52.1 to 54.0percent. Small subunit rDNA sequence analysis place this genus in thealpha subgroup of the Proteobacteria. All strains isolated in thepresent study group originated in soil. DNA reassociation studies dividethe genus into two species. K. vulgarae and K. robustum, of which K.vulgarae is the designated type species. A group of bacteria having theabove-mentioned properties does not belong to any known genera asdescribed in Bergey's Manual of Systematic Bacteriology, and thereforebelongs to a new genus.

[0035] Strain ADMX6L of Ketogulonigenium was isolated and deposited atthe Agricultural Research Service Culture Collection (NRRL), 1815 NorthUniversity Street, Peoria, Ill. 61604, USA, on Oct. 1, 1996, under theprovisions of the Budapest Treaty, and assigned accession numbers NRRLB-21627.

[0036] The present invention provides a method for conjugative transferof a vector from E. coli to Ketogulonigenium comprising culturing the E.coli with the Ketogulonigenium under such conditions such that the E.coli transfers the vector to the Ketogulonigenium. The method ofconjugative transfer relies on the ability of the vector to replicate inboth organisms, and thus requires that the vector contain replicons thatare functional in both organisms. A replicon is a nucleotide sequence,typically several hundred to several thousand base pairs long, that isvital to plasmid DNA replication. Preferably, the method comprises usingany vector that contains a replicon that is functional in E. coli, aswell a replicon that is functional in Ketogulonigenium. More preferably,the method of the invention comprises the vectors pDELIA8 and pXH2/K5.Given that the preferred method comprises using any vector that containsa replicon that is functional in E. coli, as well as a replicon that isfunctional in Ketogulonigenium, it would also be possible to transfer avector, via conjugation, from Ketogulonigenium to E. coli.

[0037] The present invention also provides a method for transformingKetogulonigenium comprising inserting a vector into the Ketogulonigeniumthrough the process of electroporation. Preferably, the vector used inelectroporation of Ketogulonigenium is pMF1014-α.

[0038] The present invention provides isolated or purified nucleic acidmolecules comprising the polynucleotides, or their complements, ofendogenous plasmids that have been isolated and purified from a strain,NRRL Deposit No. B-21627 (ADMX6L), of this novel genus. Four endogenousplasmids have been isolated from strain NRRL Deposit No. B-21627. Thenucleotide sequence shown in FIG. 1 (SEQ ID NO:1) is the nucleotidesequence of plasmid pADMX6L1 as determined by automated sequencing. Thenucleotide sequence shown in FIG. 2 (SEQ ID NO:2) is the nucleotidesequence of plasmid pADMX6L2 as determined by automated sequencing. Thenucleotide sequence shown in FIG. 3 (SEQ ID NO:3) is the nucleotidesequence of plasmid pADMX6L3 as determined by automated sequencing. Thenucleotide sequence shown in FIG. 4 (SEQ ID NO:4) is the nucleotidesequence of plasmid pADMX6L4 as determined by automated sequencing.

[0039] The endogenous plasmids contained within NRRL B-21627 (ADMX6L)have been isolated and ligated into pUC19 or pJND1000. Specifically,plasmid pADMX6L1, corresponding to SEQ ID NO:1, and pJND1000 aredigested with BamHI and ligated to each other using T4 ligase. The pXB1plasmid construct was then introduced into E. coli DH5αMCR and theculture collection was deposited at the Agricultural Research ServiceCulture Collection (NRRL), 1815 North University Street, Peoria, Ill.61604, USA, on Feb. 23, 2001, under the provisions of the BudapestTreaty, and assigned accession numbers NRRLB-30418. Plasmid pADMX6L2,corresponding to SEQ ID NO:2, and pUC19 were digested with HinDIII andligated to one another using T4 ligase. The pXH2 plasmid construct wasthen introduced into E. coli DH5αMCR and the culture collection wasdeposited at the Agricultural Research Service Culture Collection(NRRL), 1815 North University Street, Peoria, Ill. 61604, USA, on Feb.23, 2001, under the provisions of the Budapest Treaty, and assignedaccession numbers NRRL B-30419. Plasmid pADMX6L4, corresponding to SEQID NO:4, and pUC19 are digested with SspI and SmaI, respectively, andligated to one another using T4 ligase. The pXB4 plasmid construct wasthen introduced into E. coli DH5αMCR and the culture collection isdeposited at the Agricultural Research Service Culture Collection(NRRL), 1815 North University Street, Peoria, Ill. 61604, USA, on Mar.12, 2001, under the provisions of the Budapest Treaty, and assignedaccession number NRRL B-30435.

[0040] Unless otherwise indicated, all nucleotide sequences determinedby sequencing a DNA molecule herein were determined using an automatedDNA sequencer (such as the ABI Prism 3700). Therefore, as is known inthe art for any DNA sequence determined by this automated approach, anynucleotide sequence determined herein may contain some errors.Nucleotide sequences determined by automation are typically at leastabout 90% identical, more typically at least about 95% to at least about99.9% identical to the actual nucleotide sequence of the sequenced DNAmolecule. The actual sequence can be more precisely determined by otherapproaches including manual DNA sequencing methods well known in theart.

[0041] Unless otherwise indicated, each “nucleotide sequence” set forthherein is presented as a sequence of deoxyribonucleotides (abbreviatedA, G, C and T). However, by “nucleotide sequence” of a nucleic acidmolecule or polynucleotide is intended, for a DNA molecule orpolynucleotide, a sequence of deoxyribonucleotides, and for an RNAmolecule or polynucleotide, the corresponding sequence ofribonucleotides (A, G, C and U) where each thymidine deoxynucleotide (T)in the specified deoxynucleotide sequence is replaced by theribonucleotide uridine (U). For instance, reference to an RNA moleculehaving the sequence of SEQ ID NO:1 set forth using deoxyribonucleotideabbreviations is intended to indicate an RNA molecule having a sequencein which each deoxynucleotide A, G or C of SEQ ID NO:1 has been replacedby the corresponding ribonucleotide A, G or C, and each deoxynucleotideT has been replaced by a ribonucleotide U.

[0042] As indicated, nucleic acid molecules of the present invention maybe in the form of RNA, such as mRNA, or in the form of DNA, including,for instance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

[0043] By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in avector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically.

[0044] One aspect of the invention provides an isolated or purifiednucleic acid molecule comprising a polynucleotide having a nucleotidesequence selected from the group consisting of: (a) a nucleotidesequence in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, and SEQ ID NO:7; (b) a nucleotide sequence of aplasmid contained in NRRL Deposit No. B-30418, NRRL Deposit No. B-30419,and NRRL Deposit No. B-30435; and (c) a nucleotide sequencecomplementary to any of the nucleotide sequences in (a) or (b) above.

[0045] Further embodiments of the invention include isolated nucleicacid molecules that comprise a polynucleotide having a nucleotidesequence at least 90% identical, and more preferably at least 95%, 97%,98% or 99% identical, to any of the Ketogulonigenium nucleotidesequences in (a), (b) or (c) above, or a polynucleotide which hybridizesunder stringent hybridization conditions to a polynucleotide having anucleotide sequence identical to the Ketogulonigenium portion of anucleotide sequence in (a), (b) or (c), above. The polynucleotide whichhybridizes does not hybridize under stringent hybridization conditionsto a polynucleotide having a nucleotide sequence consisting of only Aresidues or of only T residues.

[0046] A polynucleotide having a nucleotide sequence at least, forexample, 95% “identical” to a reference nucleotide sequence is intendedto mean that the nucleotide sequence of the polynucleotide is identicalto the reference sequence except that the polynucleotide sequence mayinclude up to five point mutations per each 100 nucleotides of thereference nucleotide sequence. In other words, to obtain apolynucleotide having a nucleotide sequence at least 95% identical to areference nucleotide sequence, up to 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 5% of the total nucleotidesin the reference sequence may be inserted into the reference sequence.

[0047] As a practical matter, whether any particular nucleic acidmolecule is at least 90%, 95%, 97%, 98% or 99% identical to, forinstance, the nucleotide sequences shown in FIGS. 1, 2, 3, 4, 5, 6, or7, or to the nucleotide sequences of the deposited plasmids can bedetermined conventionally using known computer programs such as theFastA program. FastA does a Pearson and Lipman search for similaritybetween a query sequence and a group of sequences of the same typenucleic acid. Professor William Pearson of the University of VirginiaDepartment of Biochemistry wrote the FASTA program family (FastA,TFastA, FastX, TFastX and SSearch). In collaboration with Dr. Pearson,the programs were modified and documented for distribution with GCGVersion 6.1 by Mary Schultz and Irv Edelman, and for Versions 8 through10 by Sue Olson.

[0048] The present application is directed to nucleic acid molecules atleast 95%, 97%, 98% or 99% identical to the nucleic acid sequences shownin FIG. 1 (SEQ ID NO:1), FIG. 2 (SEQ ID NO:2), FIG. 3 (SEQ ID NO:3), andFIG. 4 (SEQ ID NO:4); or to the Ketogulonigenium portion of the nucleicacid sequence of the deposited plasmids.

[0049] The present application is also directed to nucleic acidmolecules at least 95%, 97%, 98% or 99% identical to the nucleic acidsequences shown in FIG. 5 (SEQ ID NO:5), FIG. 6 (SEQ ID NO:6), and FIG.7 (SEQ ID NO:7).

[0050] One aspect of the invention provides an isolated nucleic acidmolecule comprising a polynucleotide which hybridizes under stringenthybridization conditions to a portion of the polynucleotide of a nucleicacid molecule of the invention described above, for instance, in theplasmid contained in NRRL B-30418, NRRL B-30419, or NRRL B-30435, or inSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, or SEQ ID NO:7. By “stringent hybridization conditions” isintended overnight incubation at 42° C. in a solution comprising: 50%formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodiumphosphate (pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20μg/ml denatured, sheared salmon sperm DNA, followed by washing thefilters in 0.1×SSC at about 65° C. By a polynucleotide which hybridizesto a “portion” of a polynucleotide is intended a polynucleotide (eitherDNA or RNA) hybridizing to at least about 15 nucleotides (nt), and morepreferably at least about 20 nt, still more preferably at least about 30nt, and even more preferably about 30-70 nt of the referencepolynucleotide. These are useful as diagnostic probes and primers.

[0051] Of course, polynucleotides hybridizing to a larger portion of thereference polynucleotides (e.g., the deposited endogenous plasmids), forinstance, a portion 15-750 nt in length, or even to the entire length ofthe reference polynucleotide, are also useful as probes according to thepresent invention, as are polynucleotides corresponding to most, if notall, of the Ketogulonigenium portion of the nucleotide sequence of thedeposited DNA or the nucleotide sequence as shown in FIGS. 1 (SEQ IDNO:1), 2 (SEQ ID:NO:2), 3 (SEQ ID NO:3), 4 (SEQ ID NO:4), 5 (SEQ IDNO:5), 6 (SEQ ID NO:6), and 7 (SEQ ID NO:7). A portion of apolynucleotide of, at least 15 nt in length, for example, is intended tomean 15 or more contiguous nucleotides from the nucleotide sequence ofthe reference polynucleotide, (e.g., the deposited DNA or the nucleotidesequences as shown in FIGS. 1 (SEQ ID NO:1), 2 (SEQ ID:NO:2), 3 (SEQ IDNO:3), 4 (SEQ ID NO:4), 5 (SEQ ID NO:5), 6 (SEQ ID NO:6), 7 (SEQ IDNO:7)). As indicated, such portions are useful diagnostically either asa probe according to conventional DNA hybridization techniques or asprimers for amplification of a target sequence by the polymerase chainreaction (PCR), as described, for instance, in Molecular Cloning, ALaboratory Manual, 2nd. edition, edited by Sambrook, J., Fritsch, E. F.and Maniatis, T., (1989), Cold Spring Harbor Laboratory Press, theentire disclosure of which is hereby incorporated herein by reference.

[0052] One embodiment of the present invention is a vector comprisingthe nucleic acid molecules contained in the group consisting of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, corresponding toendogenous plasmids contained in Deposit No. NRRL B-21627 (ADMX6L). Apreferred embodiment of the present invention is that polynucleotides ofinterest can be joined to the nucleic acid molecules of the presentinvention, which may contain a selectable marker. An additionalpreferred embodiment is that the vectors comprising the nucleic acidmolecules also contain a transcription terminator, a promoter and apolylinker site. As used herein, a polylinker site is defined to be adiscrete series of restriction endonuclease sites that occur between thepromoter and the terminator. The vector can optionally contain itsnative expression vector and/or expression vectors which includechromosomal-, and episomal-derived vectors, e.g., vectors derived frombacterial exogenous plasmids, bacteriophage, and vectors derived fromcombinations thereof, such as cosmids and phagemids.

[0053] A DNA insert of interest should be operatively linked to anappropriate promoter, such as its native promoter or a host-derivedpromoter, the phage lambda P_(L) promoter, the phage lambda P_(R)promoter, the E. coli lac promoters, such as the lacI and lacZpromoters, trp and tac promoters, the T3 and T7 promoters and the gptpromoter to name a few. Other suitable promoters will be known to theskilled artisan.

[0054] The expression constructs will preferably contain sites fortranscription initiation, termination and, in the transcribed region, aribosome binding site for translation. The coding portion of the maturetranscripts expressed by the constructs can include a translationinitiating codon at the beginning and a termination codon appropriatelypositioned at the end of the coding sequence to be translated.

[0055] As indicated, the expression vectors will preferably include atleast one marker capable of being selected or screened for. Preferablythe selectable marker comprises a nucleotide sequence which confersantibiotic resistance in a host cell population. Such markers includeamikacin, ampicillin, chloramphenicol, erythromycin, gentamicin,kanamycin, penicillin, spectinomycin, streptomycin, or tetracyclineresistance genes. Other suitable markers will be readily apparent to theskilled artisan.

[0056] The translated polypeptide encoded by the DNA in the vector maybe expressed in a modified form, such as a fusion protein, and mayinclude not only secretion signals, but also additional heterologousfunctional regions. Thus, for instance, a region of additional aminoacids, particularly charged amino acids, may be added to the N-terminusof the polypeptide to improve stability and persistence in the hostcell, during purification, or during subsequent handling and storage.Also, peptide moieties may be added to the polypeptide to facilitatepurification.

[0057] The translated protein encoded by the DNA contained in the vectorcan be recovered and purified from recombinant cell cultures bywell-known methods including ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification.

[0058] The present invention also provides vectors comprising nucleicacid molecules comprising a nucleotide sequence of a replicon found onan endogenous plasmid contained in Deposit No. NRRL B-21627. Preferably,the vector of the present invention comprises a replicon selected fromthe group of nucleotide sequences comprising SEQ ID NO:5, SEQ ID NO:6and SEQ ID NO:7. The replicon is a nucleotide sequence that is typicallyseveral hundred to several thousand base pairs long and encodesfunctions controlling the replication of plasmid DNA. The nucleotidesequence in FIG. 5 (SEQ ID NO:5) is the nucleotide sequence comprising areplicon on plasmid pADMX6L1. The nucleotide sequence in FIG. 6 (SEQ IDNO:6) is the nucleotide sequence comprising a replicon on plasmidpADMX6L2. The nucleotide sequence in FIG. 7 (SEQ ID NO:7) is thenucleotide sequence comprising a replicon on plasmid pADMX6L3.

[0059] The present invention further provides a vector which has anreplicon that is functional in Escherichia coli (E. coli) as well as areplicon that is functional in Ketogulonigenium. The present inventionalso provides a vector which has a replicon that is functional inKetogulonigenium, as well as a replicon that is functional in any of thegenera comprising the group consisting of Acetobacter, Corynebacterium,Rhodobacter, Paracoccus, Roseobacter, Pseudomonas, Pseudogluconobacter,Gluconobacter, Serratia, Mycobacterium, Streptomyces and Bacillus.Utilizing the fact that the vector comprises a functional replicon inKetogulonigenium, and preferably also comprises a replicon functional inE. coli, the present invention provides for a transformed cell of thegenus Ketogulonigenium, comprising the vector. The present inventionalso provides for a transformed E. coli, comprising the vector. E. coliis known to be an efficient host for amplification of a vector DNA andmanipulation of recombinant DNA by simple and rapid methods. On theother hand, Ketogulonigenium can be used as a host for expression ofKetogulonigenium genes. Since the vectors of the present invention aresuch functional constructs, they enable cloning of certain genes ofKetogulonigenium in E. coli and thereafter the effective expression ofthe genes in Ketogulonigenium. Furthermore, it is favorable that suchfunctional constructs also contain a DNA region necessary for conjugaltransfer (mob site). Hence the vectors of the present invention canfirst be assembled in E. coli and then directly introduced intoKetogulonigenium by conjugal mating without isolation of plasmid DNAfrom E. coli.

[0060] The present invention further relates to (a) nucleic acidsequences comprising at least 95% functional homology with thoseencoding polypeptides derived from the four endogenous plasmids fromKetogulonigenium strain ADMX6L, and (b) to amino acid sequencescomprising at least 95% functional homology with those encoded withinthe plasmids. The invention also relates to nucleic acid sequencescomprising at least 95% functional homology with, pADMX6L1 rep ORF(bases 2255-2710 of FIG. 1 (SEQ ID NO:1), pADMX6L2 rep ORF (reversecompliment of bases 3960-2562 of FIG. 2 (SEQ ID NO:2), and pADMX6L3 repORF (reverse compliment of bases 5031-4003 of FIG. 3 (SEQ ID NO:3)). Theinvention further relates to amino acid sequences comprising at least95% functional homology with those set forth in FIGS. 8, 9 or 10.

[0061] Methods used and described herein are well known in the art andare more particularly described, for example, in R. F. Schleif and P. C.Wensink, Practical Methods in Molecular Biology, Springer-Verlag (1981);J. H. Miller, Experiments in Molecular Genetics, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1972); J. H. Miller, A ShortCourse in Bacterial Genetics, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1992); M. Singer and P. Berg, Genes & Genomes,University Science Books, Mill Valley, Calif. (1991); J. Sambrook, E. F.Fritsch and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2d ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); P.B. Kaufman et al., Handbook of Molecular and Cellular Methods in Biologyand Medicine, CRC Press, Boca Raton, Fla. (1995); Methods in PlantMolecular Biology and Biotechnology, B. R. Glick and J. E. Thompson,eds., CRC Press, Boca Raton, Fla. (1993); P. F. Smith-Keary, MolecularGenetics of Escherichia coli, The Guilford Press, New York, N.Y. (1989);Plasmids: A Practical Approach, 2nd Edition, Hardy, K. D., ed., OxfordUniversity Press, New York, N.Y. (1993); Vectors: Essential Data,Gacesa, P., and Ramji, D. P., eds., John Wiley & Sons Pub., New York,N.Y. (1994); Guide to Electroporation and electrofusions, Chang, D., etal., eds., Academic Press, San Diego, Calif. (1992); PromiscuousPlasmids of Gram-Negative Bacteria, Thomas, C. M., ed., Academic Press,London (1989); The Biology of Plasmids, Summers, D. K., BlackwellScience, Cambridge, Mass. (1996); Understanding DNA and Gene Cloning: AGuide for the Curious, Drlica, K., ed., John Wiley and Sons Pub., NewYork, N.Y. (1997); Vectors: A Survey of Molecular Cloning Vectors andTheir Uses, Rodriguez, R. L., et al., eds., Butterworth, Boston, Mass.(1988); Bacterial Conjugation, Clewell, D. B., ed., Plenum Press, NewYork, N.Y. (1993); Del Solar, G., et al., “Replication and control ofcircular bacterial plasmids,” Microbiol. Mol. Biol. Rev. 62:434-464(1998); Meijer, W. J., et al., “Rolling-circle plasmids from Bacillussubtilis: complete nucleotide sequences and analyses of genes ofpTA1015, pTA1040, pTA1050 and pTA1060, and comparisons with relatedplasmids from gram-positive bacteria,” FEMS Microbiol. Rev. 21:337-368(1998); Khan, S. A., “Rolling-circle replication of bacterial plasmids,”Microbiol. Mol. Biol. Rev. 61:442-455 (1997); Baker, R. L., “Proteinexpression using ubiquitin fusion and cleavage,” Curr. Opin. Biotechnol.7:541-546 (1996); Makrides, S. C., “Strategies for achieving high-levelexpression of genes in Escherichia coli,” Microbiol. Rev. 60:512-538(1996); Alonso, J. C., et al., “Site-specific recombination ingram-positive theta-replicating plasmids,” FEMS Microbiol. Lett.142:1-10 (1996); Miroux, B., et al., “Over-production of protein inEscherichia coli: mutant hosts that allow synthesis of some membraneprotein and globular protein at high levels,” J. Mol. Biol. 260:289-298(1996); Kurland, C. G., and Dong, H., “Bacterial growth inhibited byoverproduction of protein,” Mol. Microbiol. 21:1-4 (1996); Saki, H., andKomano, T., “DNA replication of IncQ broad-host-range plasmids ingram-negative bacteria,” Biosci. Biotechnol. Biochem. 60:377-382 (1996);Deb, J. K., and Nath, N., “Plasmids of corynebacteria,” FEMS Microbiol.Lett. 175:11-20 (1999); Smith, G. P., “Filamentous phages as cloningvectors,” Biotechnol. 10:61-83 (1988); Espinosa, M., et al., “Plasmidrolling cicle replication and its control,” FEMS Microbiol. Lett.130:111-120 (1995); Lanka, E., and Wilkins, B. M., “DNA processingreaction in bacterial conjugation,” Ann. Rev. Biochem. 64:141-169(!995); Dreiseikelmann, B., “Translocation of DNA across bacterialmembranes,” Microbiol. Rev. 58:293-316 (1994); Nordstrom, K., andWagner, E. G., “Kinetic aspects of control of plasmid replication byantisense RNA,” Trends Biochem. Sci. 19:294-300 (1994); Frost, L. S., etal., “Analysis of the sequence gene products of the transfer region ofthe F sex factor,” Microbiol. Rev. 58:162-210 (1994); Drury, L.,“Transformation of bacteria by electroporation,” Methods Mol. Biol.58:249-256 (1996); Dower, W. J., “Electroporation of bacteria: a generalapproach to genetic transformation,” Genet. Eng. 12:275-295 (1990); Na,S., et al., “The factors affecting transformation efficiency ofcoryneform bacteria by electroporation,” Chin. J. Biotechnol. 11:193-198(1995); Pansegrau, W., “Covalent association of the traI gene product ofplasmid RP4 with the 5′-terminal nucleotide at the relaxation nicksite,” J. Biol. Chem. 265:10637-10644 (1990); and Bailey, J. E.,“Host-vector interactions in Escherichia coli,” Adv. Biochem. Eng.Biotechnol. 48:29-52 (1993).

EXAMPLES

[0062] The following examples are illustrative only and are not intendedto limit the scope of the invention as defined by the appended claims.

Example 1

[0063] Preparation of Plasmid DNA from Ketogulonigenium and E. coliStrains

[0064] A portion of a frozen culture of Ketogulonigenium robustum ADMX6L(NRRL B-12627) was seeded into 10 ml X6L medium (2% mannitol, 1% yeastextract, 1% soytone, 0.5% malt extract, 0.5% NaCl, 0.25% K₂HPO₄, pH 7.8)and grown overnight at 30° C. A portion of a frozen culture ofEscherichia coli transformed with pJND1000 was seeded into 10 ml ofLuria Broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl) and grownovernight at 37° C. Both cultures were used to prepare plasmid DNA.

[0065] DNA was isolated using the Promega Wizard Plus Midipreps DNAPurification System (Madison, Wis.). The culture was centrifuged at10,000×g for 10 minutes at 4° C. in a Sorval RC-5B centrifuge using theSS-34 rotor. The pellet was suspend in 3 ml of 50 mM Tris-HCl, pH 7.5,10 mM EDTA, 100 μg/ml RnaseA. Three ml of cell lysis solution (0.2MNaOH, 1% SDS) was added and mixed by inverting the tube, then three mlof neutralization solution (1.32M potassium acetate, pH4.8) was addedand mixed by inverting the tube. The lysate was centrifuged at 14,000×gfor 15 minutes at 4° C. in a SS-34 rotor, then the supernatant wascarefully decanted to a new centrifuge tube. Ten ml of resuspensionresin (40% isopropanol, 4.2M guanidine hydrochloride) was added to thesupernatant fluid and mixed by swirling, then the mixture wastransferred into the Promega Wizard Midicolumn, which was connected avacuum manifold. Vacuum was applied to pull the resin/DNA mixturecompletely into the midicolumn. The column was washed twice with 15 mlof column wash solution (95% ethanol, 80 mM potassium acetate, 8.3 mMTris-HCl, pH 7.5, 40 μM EDTA. The reservoir was removed from themidicolumn with a scissors, then the column was placed in amicrocentrifuge tube and centrifuged at 10,000×g for 2 minutes to removeany residual solution. The midicolumn was transferred to a newmicrocentrifuge tube and 300 μl of sterile dH₂O was applied. The tubewas microcentrifuged at 10,000×g for 20 seconds to elute to the DNA intosolution. A similar procedure would be employed for other plasmids fromKetogulonigenium or E. coli, except that choice and concentration ofselective antibiotics would be altered as suitable for the plasmid beingisolated.

Example 2 Transformation of a Ketogulonigenium Host with a Plasmid UsingElectroporation

[0066]Ketoguonigenium robustum strain ADMX6L (NRRL B-21627) wastransformed with plasmid pMF1014-α using the electroporation method.Plasmid pMF1014-α was described in a Ph.D. thesis at MIT (M. T.Follettie, “DNA Technology for Corynebacterium glutamicum: Isolation andCharacterization of Amino Acid Biosynthetic Genes,” Ph.D. Dissertation,Massachusetts Institute of Technology, Cambridge, Mass. (1989)).pMFW1014α encodes kanamycin resistance and can replicate and bemaintained in both E. coli and in Ketogulonigenium.

[0067] Competent Ketogulonigenium cells were prepared by seeding asingle colony of ADMX6L into 10 ml of X6L medium (1% soytone, 1% yeastextract, 0.5% malt extract, 0.5% NaCl, 0.25% K₂HPO₄, 2% mannitol, pH7.8). The culture was shaken at 300 rpm at 30 C until reaching anoptical density of 0.8 units at 600 nm wavelength. Five ml of thisculture was used to seed 500 ml of fresh X6L medium in a 2 L bafflederlenmeyer flask, which was shaken at 300 rpm at 30° C. until reachingan optical density of 0.8 units. The culture was chilled in an ice-waterbath 10 minutes, then transferred to a pre-chilled centrifuge bottle andcentrifuged at 5,000 rpm in a Sorvall 5C-RB Refrigerated Centrifuge for15 minutes at 4° C. Cells were maintained at 2-4° C. for all steps tofollow. The supernatent was decanted and the cell pellet suspended in 5ml ice-cold Milli-Q water, then additional cold Milli-Q water was wasadded to a volume of 500 ml. The cells were centrifuged as before, thenrewashed in 500 ml of Milli-Q water as before and recentrifuged. Thetwice-washed cell pellet was suspended in 40 ml of ice-cold 10% glycerolthen centrifuged again as before. The supernatent was decanted, then thecells were suspended in a volume of chilled 10% glycerol approximatelyequal to the volume of the cell pellet. The competent Ketogulonigeniumcells were aliquoted to microcentrifuge tubes (40 μl per tube) andstored at −80° C.

[0068] A BioRad “Gene Pulser II” electroporator device was set to 1.5kV, 25 uF. The pulse controller was set to 200 Ohms. One μl of pMF1014-αDNA prepared as in Example 1 was added to 40 μl of thawed chilledcompetent Ketogulonigenium cells on ice. The cell-DNA mixture wastransferred to a pre-chilled electroporation cuvette, which was thentransferred to the electroporation device and the pulse was applied. Oneml of X6L medium was added to the cuvette, then the mixture was removed,transferred to a 10-ml test tube, and incubated for 2 hours with orbitalshaking at 300 rpm and 30° C. The incubated cells (approximately 1.4 ml)were placed in a microcentrifuge tube and spun at 13,000 rpm for 2minutes, after which 0.9 ml of clear supernatent was carefully removed.The cell pellet was suspended in the remaining supernatent, then thecells were spread onto the surface of an X6L medium agar plate (1.2%Difco Bacto Agar) containing 50 μg/ml of kanamycin, and the plate wasincubated for 2-3 days at 30° C. Colonies that formed on this plate wereKetogulonigenium transformed with plasmid vector pMF1014-α.

Example 3 Transfer of Plasmids From E. coli to Ketogulonigenium, DepositNo. NRRL B-21627 by Bi-parental or Tri-parental Mating

[0069] (A) Bi-parental Mating

[0070] Plasmid pDELIA8 was transferred into Ketogulonigenium from E.coli by conjugation. To make pDELIA8, the AflIII-SphI fragment fromplasmid pFD288 (GenBank Accession No. U30830), which contains the clonedmob region of plasmid RK2 (GenBank Accession No. L27758), was isolatedfrom agarose gels and ligated to the large AflIII-SphI fragment fromplasmid pUC19 (GenBank Accession No. M77789) to make the intermediateplasmid pUC19/oriT. pUC19/oriT DNA was digested with SspI and DraI andthe large (2527 bp) fragment was purified from an agarose gel. PlasmidpMF1014-α (M. T. Follettie, “DNA Technology for Corynebacteriumglutamicum: Isolation and Characterization of Amino Acid BiosyntheticGenes”, Ph.D. Dissertation, Massachusetts Institute of Technology,Cambridge, Mass. (1989)) was digested with BamHI and PstI, made bluntended with Klenow fragment, and the large fragment was isolated from anagarose gel. The gel purified, blunt-ended fragments from pUC19/oriT andpMF1014-α were ligated together to make pDELIA8. pDELIA8 carries a genefor kanamycin resistance, a plasmid replication gene, the replicon thatis functional in E. coli and in Ketogulonigenium, lacZα and apolylinker.

[0071] A 5 ml culture of E. coli S17-1 (ATCC 47055) was transformed withpDELIA8 using calcium chloride mediated transformation. TheS17-7/pDELIA8 strain was grown in Luria Broth containing 50 μg/mlkanamycin at 37° C. overnight. Ketogulonigenium robustum strain ADMX6L01was grown in X6L medium containing 50 μg/ml of nalidixic acid at 30° C.overnight. ADMX6L01 is a nalidixic acid resistant mutant of strainADMX6L, derived by conventional mutagenesis of ADMX6L followed byselection of strains showing resistance to 50 μg/ml of nalidixic acid.

[0072] Fifty microliters of the fresh S17-1/pDELIA8 culture wastransferred into 3 ml of Luria Broth containing 50 μg/ml of kanamycinand 5 μg/ml of tetracycline and grown at 37° C. until reaching an OD₆₀₀of 0.2 to 0.4 absorption units at 600 nm. Two hundred microliters of thefresh ADMX6L01 culture was transferred into 3 ml of X6L mediumcontaining nalidixic acid and grown at 30° C. until reaching an OD₆₀₀ of0.8 absorption units. 400 μl of S17-1/pDELIA8 culture was placed in a1.5 ml microcentrifuge tube and microcentrifuged for 2 minutes. Thepellet was resuspended in X6L medium without antibiotics and spun downagain. This pellet was resuspended in 1 ml of the fresh ADMX6L01 cultureto achieve a suspension of mixed cells. The mixed cells were centrifugedto a pellet, resuspended in 1 ml of fresh X6L medium withoutantibiotics, then pelleted and washed once again in 1 ml of X6L medium.The final pellet of mixed cells was then resuspended in 100 μl of X6Lmedium without antibiotics and spotted onto a a sterilized GN-6 metrical0.45 μm×25 mm filter (Gelman Sciences, Product No. 63068) resting on thesurface of an X6L agar medium plate without antibiotics, which was thenincubated overnight at 30° C. The cell biomass on the filter wasresuspended in 3 ml of X6L medium and 50 μl of this suspension wasplated onto X6L agar medium containing kanamycin and nalidixic acid atconcentration 50 μg/ml each. Colonies that formed on this plate after2-3 days incubation at 30° C. were Ketogulonigenium transconjugantstransformed with pDELIA8.

[0073] (B) Tri-parental Mating

[0074] A 5 ml culture of E. coli HB101 (ATCC33694) harboring plasmidpDELIA8 was grown in Luria Broth (LB) containing 50 μg/ml of kanamycinat 37 degrees overnight. A 5 ml culture of E. coli HB101 harboringplasmid RP1 (GenBank Accession No. L27758) was grown in Luria Brothcontaining 5 μg/ml of tetracycline at 37 degrees overnight. A 5 mlculture of Ketogulonigenium robustum ADMX6L01 was grown in X6L mediumcontaining 50 μg/ml of nalidixic acid at 30 degrees overnight. Fiftymicroliters each of the fresh E. coli HB101/pDELIA8 and HB101/RP1cultures were transferred to 3 ml of LB with 50 μg/ml of kanamycin or 5μg/ml of tetracycline, respectively, and were grown to an OD_(600 nm) of0.2 to 0.4. Two hundred microliters of the fresh Ketogulonigeniumrobustum ADMX6L01 culture was transferred to 3 ml of X6L mediumcontaining 50 μg/ml of nalidixic acid and grown to an OD_(600 nm) of0.8. Four hundred microliters each of the E. coli cultures was combinedand pelleted in a 1.5 ml microfuge tube, then decanted, resuspended in 1ml of X6L medium without antibiotics and spun down again. Thesupernatant was removed and the E. coli pellet was resuspended in 1 mlof the fresh ADMX6L01 culture. The three-strain mixture was centrifugedto a pellet, then resuspended again 1 ml of X6L medium withoutantibiotics and recentrifuged. The pellet from this wash was resuspendedin 100 μl of X6L medium without antibiotics and spotted onto asterilized GN-6 metrical 0.45 μm×25 mm filter (Gelman Sciences, ProductNo. 63068) resting on the surface of an X6L agar medium plate withoutantibiotics, which was then incubated overnight at 30° C. The cellbiomass on the filter was resuspended in 3 ml of X6L medium and 50 μl ofthis suspension was plated onto X6L agar medium containing kanamycin andnalidixic acid at concentration 50 μg/ml each. Colonies that formed onthis plate after 2-3 days incubation at 30° C. were Ketogulonigeniumtransconjugants transformed with plasmid pDELIA8.

Example 4 Construction of Plasmid Vector pJND1000

[0075] pJND1000 was constructed from segments of plasmids pUC19 (GenBankAccession No. M77789), pUC4K (GenBank Accession No. X06404), pFD288(GenBank Accession No. U30830), and pFC5 (David M. Lonsdale et. al.,“pFC1 to pFC7: A novel family of combinatorial cloning vectors.”, PlantMolecular Biology Reporter 13(4):343-345 (1995)).

[0076] The ampicillin resistance gene (amp^(R)) was removed from pUC19by digesting pUC19 DNA with restriction enzymes DraI and SspI,separating the 1748 bp vector fragment from the smaller amp^(R) fragmentand other fragments by gel electrophoresis, then recovering the 1748 bpfragment from a gel slice. A kanamycin resistance gene (kan^(R))fragment from pUC4K was prepared by digesting pUC4K with restrictionenzyme PstI, treating the mixture with Klenow Fragment to produce bluntends, separating the fragments by gel electrophoresis, then purifyingthe 1240 bp kan^(R) fragment from a gel slice. The isolated fragmentsfrom pUC19 and pUC4K were mixed and ligated with T4 ligase following theprotocol of GibcoBRL technical bulletin 15244-2 (Rockville, Md.) toproduce “intermediate p1”, an intermediate plasmid carrying pUC19features and a kan^(R) gene. Strain E. coli DH5αMCR was transformed withthe ligation mixture following an established protocol (“Fresh CompetentE. coli prepared using CaCl₂”, pp. 1.82-1.84, in J. Sambrook, E. F.Fritsch and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Ed.(1989)). Transformants were selected on Luria Broth plates containing 50μg/ml of kanamycin.

[0077] An oriT site for conjugative transfer was obtained from plasmidpFD288 by restricting it with HaeII, converting the single stranded endsto blunt ends by treating the mixture with Klenow Fragment, separatingthe fragments by agarose gel electrophoresis, then purifying the 778 bporiT fragment from a gel slice. The intermediate p1 plasmid was openedat a single site with restriction enzyme SapI, then treated with KlenowFragment to convert the single stranded ends to blunt ends. The SapIdigested, blunt ended p1 intermediate was mixed with the purified oriTfragment, then treated with T4 ligase as above to create “intermediatep2”, a plasmid which carries oriT in addition to kan^(R) and otherpUC19-derived features. Strain E. coli DH5αMCR was transformed with theligation mixture as above. Intermediate p2 was confirmed by restrictiondigestion analysis.

[0078] The polylinker in intermediate plasmid p2 was replaced with thepolylinker from pFC5. To do this the two plasmids were separatelyrestricted with PvuII, the fragments from each digestion reaction wereseparated by gel electrophoresis, then the pFC5-derived polylinkerfragment (531 bp), and the larger non-polylinker fragment fromintermediate p2 were purified from gel slices. The recovered p2 fragmentand the pFC5-derived polylinker fragment were mixed and joined using T4ligase as above to make plasmid pJND1000. The structure of pJND1000 wasconfirmed by restriction digestion analysis. PJND1000 replicates in E.coli, has a functioning kanamycin resistance gene, an RK2-derived oriTsite to permit conjugative transfer into Ketogulonigenium and otherhosts, a polylinker for DNA cloning, forward and reverse M13 primers tofacilitate DNA sequencing reactions into the polylinker, and permitsscreening for cloned inserts by inactivation of lacZα using Xgalindicator plates.

Example 5 Isolation of Plasmid pXB1 Containing LinearizedKetogulonigenium Plasmid pADMX6L1

[0079] The DNA sequence of plasmid pADMX6L1 (SEQ ID NO: 1) is about 7029bp long and contains a single BamHI restriction site. The BamHI site wasutilized to clone the pADMX6L1 sequence into the E. coli vectorpJND1000. A DNA prep containing a mixture of the endogenous plasmidsfrom Ketogulonigenium robustum ADMX6L, and purified pJND1000 DNA from E.coli, were made as in Example A and separately digested with restrictionenzyme BamHI. A Wizard DNA Clean-Up kit was used to separate thedigested DNA from enzyme and salts in preparation for the next step,with a final DNA suspension volume of 50 μl. DNA from the two digestions(3 μl of pJND1000 DNA and 10 μl of Ketogulonigenium plasmid DNA) wasmixed and ligated overnight at room temperature using T4 ligase with theprotocol of GibcoBRL technical bulletin 15244-2 (Rockville, Md.). StrainE. coli DH5αMCR was transformed with the ligation mixture following anestablished protocol (“Fresh Competent E. coli prepared using CaCl₂”,pp. 1.82-1.84, in J. Sambrook, E. F. Fritsch and T. Maniatis, MolecularCloning: A Laboratory Manual, 2nd Ed. (1989)). Transformants were spreadon Luria Broth agar plates containing 50 μg/ml of kanamycin and 40 μg/mlof Xgal and grown at 37 deg C. Colonies that were white, indicatinginsertion of DNA into the pJND1000 BamHI site, were picked and culturedfor further processing. Digestion of plasmid DNA from transformantsusing BamHI, EcoRV, and XhoI separately, yielded the expected fragmentsizes for sucessful cloining of pADMX6L1 into the pJND1000 E. colivector. The identity of the pADMX6L1 insert was further confirmed bypartial DNA sequencing into the pADMX6L1 DNA region using the M13sequencing primer regions of the pJND1000 vector. The chimeric plasmidcontaining linearized pADMX6L1 cloned into pJND1000 was named pXB1. AnE. coli host transformed with pXB1 was deposited in the patentcollection of the National Regional Research Laboratories in Peoria,Ill., U.S.A. under the terms of the Budapest Treaty as NRRL B-30418.

Example 6 Isolation of Plasmid pXH2 Containing LinearizedKetogulonigenium Plasmid pADMX6L2

[0080] The DNA sequence of plasmid pADMX6L2 (SEQ ID NO: 2) is about 4005bp long and contains a single HinDIII restriction site. The HinDIII sitewas utilized to clone the pADMX6L2 sequence into the E. coli vectorpUC19. The procedure was the same as in Example 5, except that prior tothe ligation step the plasmid DNAs were digested with HinDIII instead ofwith BamHI, and transformants were plated on LB agar plates containingXgal and 100 μg/ml of ampicillin instead of the kanamycin. Plasmid DNAfrom transformants giving white colonies was isolated and digested withHinDIII and NdeI, giving the expected fragment sizes for correctinsertion of linearized pADMX6L2 DNA into the pUC19 vector. The identityof the pADMX6L2 insert was further confirmed by partial DNA sequencinginto the pADMX6L2 region using the M13 primer regions of pUC19. Thechimeric plasmid containing linearized pADMX6L2 cloned into pUC19 wasnamed pXH2. An E. coli host transformed with pXH2 was deposited in thepatent collection of the National Regional Research Laboratories inPeoria, Ill., U.S.A. under the terms of the Budapest Treaty as NRRLB-30419.

Example 7 Construction of E. coli/Ketogulonigenium Shuttle PlasmidpXH2/K5

[0081] Since kanamycin resistance can be expressed in Ketogulonigenium,it was desireable to replace the ampR resistance gene in pXH2 with akanamycin resistance gene, thereby allowing the selective isolation ofKetogulonigenium strains transformed with a plasmid. In vitrotransposition was used to move a kanamycin resistance gene into pXH2 andsimultaneously inactivate ampicillin resistance. Insertion of akanamycin resistance gene into the ampicillin resistance gene of pXH2was achieved using Epicentre technologies EZ::TN Insertion System(Madison, Wis.). 0.05 pmoles of pXH2 was combined with 0.05 pmoles ofthe <KAN-1> Transposon, 1 μl of EZ::TN 10×Reaction Buffer, 1 μl ofEZ::TN Transposase, and 4 ul of sterile water giving a total volume of10 μl in the transposition reaction. This mixture was incubated at 37°C. for 2 hours, then stopped by adding 1 μl of EZ::TN 10×Stop Solution,mixing, and incubation at 70° C. in a heat block for 10 minutes. E. coliDH5αMCR was transformed with the DNA mixture and transformants wereselected on Luria Broth agar plates containing 50 μg/ml of kanamycin.Colonies recovered from these plates were patched onto two LB agarplates, one with 50 μg/ml of kanamycin and the other with 100 μg/ml ofampicillin. Only those that grew on the kanamycin plates were saved.Plasmid DNA from an ampicillin sensitive, kanamycin resistant colony wasisolated as “pXH2/K5”. The proper insertion of the kanamycin resistancetransposon into the vector ampicillin resistance gene was confirmed byanalyzing pXH2/K2 DNA with various restriction endonucleases. Todemonstrate the viability of pXH2/K5 as an E. coli/Ketogulonigeniumshuttle vector, Ketogulonigenium robustum strain ADMX6L was transformedwith pXH2/K5 DNA using the electroporation technique of Example 2.Stable, kanamycin resistant transformants were obtained from whichpXH2/K5 DNA can be reisolated.

Example 8 Isolation of Plasmid pXB4 Containing LinearizedKetogulonigenium Plasmid pADMX6L4

[0082] The DNA sequence of plasmid pADMX6L4 (SEQ ID NO: 4) is about 4005bp long and contains a single SspI restriction site. The SspI site wasutilized to clone the pADMX6L2 plasmid into the E. coli vector pUC19.The procedure was the same as in Example 6, except that prior to theligation step the plasmid DNA from strain ADMX6L was digested with SspIand pUC19 was digested with SmaI. Plasmid DNA from transformants givingwhite colonies was isolated and double digested with EcoRI and BamHI,and separately with PstI and SphI, in each case giving the expectedfragment sizes for correct insertion of linearized pADMX6L4 DNA intopUC19. Partial DNA sequencing into the pADMX6L4 region using the M13primers of pUC19 further confirmed the identity of the pADMX6L4 insert.The chimeric plasmid containing linearized pADMX6L4 cloned into pUC19was named pXB4. An E. coli host transformed with pXB4 was deposited inthe patent collection of the National Regional Research Laboratories inPeoria, Ill., U.S.A. under the terms of the Budapest Treaty as NRRLB-30435.

Example 9 Definition of DNA Sequences Comprising Replication Functionsof Ketogulonigenium Plasmids

[0083] Various programs of the Wisconsin Package version 10.1 (GeneticsComputer Group, Inc. Madison, Wis.) were used to analyze the DNAsequences of the four endogenous plasmids from Ketogulonigenium strainADMX6L. The analysis revealed multiple open reading frames (ORFs),nucleotide sequences having protein-encoding potential, on each plasmid.The predicted amino acid sequences of these ORFs was obtained, and asimilarity search against PIR and SWISS-PROT protein databases wasconducted using the GCG implementations of the FASTA (Proc. Natl. Acad.Sci. USA 85:2444-2448 (1988)) and BLAST (Altschul et al., Nucleic AcidsResearch 25:3389-3402 (1997)) methods. Based on sequence similarity toknown plasmid-encoded replication proteins, plasmids pADMX6L1, pADMX6L2,and pADMX6L3 were found to encode potential plasmid replicationproteins. The genes (ORFs) for these functions have the followingendpoints: pADMX6L1 rep ORF: bases 2255-2710 of FIG. 1 (SEQ ID NO:1)pADMX6L2 rep ORE: reverse compliment of bases 3960-2562 of FIG. 2 (SEQID NO:2) pADMX6L3 rep ORE: reverse compliment of bases 5031-4003 of FIG.3 (SEQ ID NO:3).

[0084] A region of DNA sequence upstream and downstream of the regionsdefined by these ORFs, perhaps 500 bp in each direction, is likely tocontain transcriptional promoters, terminators, and other sequencesrequired for proper expression of the replication proteins and controlof plasmid replication. Therefore a region comprising part or all of theplasmid replicon for these three plasmids could be defined by thefollowing DNA sequences: pADMX6L1 replicon: bases 1755-3210 of the FIG.1 sequence, also represented by SEQ ID NO:5 pADMX6L2 replicon: bases455-2060 of the FIG. 2 sequence, also represented by SEQ ID NO:6pADMX6L3 replicon: bases 3503-5531 of the FIG. 3 sequence, alsorepresented by SEQ ID NO:7

[0085] All publications mentioned herein above are hereby incorporatedin their entirety by reference. All publications cited in theannotations to the Genbank accession numbers or in ATCC straindescriptions cited herein are hereby incorporated in their entirety byreference.

[0086] While the foregoing invention has been described in some detailfor purposes of clarity and understanding, it will be appreciated by oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention and appended claims.

1 10 1 7029 DNA Ketogulonigenium misc_feature pADMX6L1 1 gccatttctgcgctgcactt cgctaagggt tcaagggaaa cgcagggttc ccttgcccac 60 acaaacgcgcagcgtttgta taagtgggca cttcgtgttt gacacgctat ccactacgcg 120 gcacaaattcaactcttgta acgaggaagg gcggtagaat ggcgcgcacc atcgaccagc 180 agatcgcagatgcgcaagcg aagctggcgc ggctcaaaac ccgtcagaaa gccagcgaca 240 cccgccgaaagatcatcgtc ggcgccatcg tcaccaccga ggccctgaaa gaccccaaga 300 tttccaaatggctggcatct accctgcgca agaacgcaac ccgggacgtg gaccagaagg 360 aaatcgccgggctgctggcc gacctcgatg ccagggcgca aagcgccggg gcgggtgagg 420 catgagcggcagcaccgatc cgtttctggt tctggtcgat gatattggcg cgctgcgccg 480 ccagatcgagaacctgcaac gcaccagcct cgacagggac gaggccgaac atctcaacgc 540 gaccatcgcccagagcctcg acaacatggc gcaaaccgga aaacggctgg aacagcgcct 600 tgagggccagttgcagctcg ccaccgccaa aacccacagg gacgccatag aagccgctca 660 gggggccgccagagcggcta tcagggaatc ccatgccgag atcctccaaa cggccaggag 720 cctctcacaggccgcaggag aggcccgcag agaggcgtgg cgctggttcg gcgggttctg 780 ggtctggctggcctcgatcg gggccgcagg ggcgcttgtc ggcgcgctgg ccgtgttctg 840 gctccagggccgcgccgatg ccaaagcctt cggacagtat cccagcatct actgcaccac 900 cgcaggcggggcattcgccg atcagcgcga cggaagccga tactgcatct tcatgatttc 960 accgccgacacagccagacg gggaatgacg gcttacgcgc cgggctggat cgagactttc 1020 agcccgagcgccttagcgac cttcatcacc gtggacagcg tagggttccc atccccggat 1080 agcgccttgttcagccccac ccggctcatg ccaacctcac gggccagcgc ggtcatgttc 1140 cgtgcgcgggcaaccactcc aagggcgcgg gcaacatagg cgggatcgtc gccgccatct 1200 tccatgaccgcttcgagata ggccgcaata tcttcctcgg tcttgaggta gtcggcggaa 1260 tcgtagcgggcgaatttttc ttccggcatc gcttagccct tccactctgc ggccagcacc 1320 ttggcctgtttgatgtcttt gctctgcgtg gacttgtcgc cgccacaaag caggatcacg 1380 agaaccggcccgcgctggat gaaatacacc cggtagcccg gcccgtagtt gatccgcagt 1440 tccgaaacaccctctccgac cggctccaca tcgccggggt tccccgccgc aaggcggtcc 1500 agtctggcagtgatgcgcgc aaccgccctg cgatcccgca aaccggaaag ccaggtatcg 1560 aaggttccgcttcggattaa ctcgatcatt cgacaactat agttatcatg tgggtgtctg 1620 acaaccagagttatcacttc cttgttctaa gcaaatccga ggccagccac gggcgtagcc 1680 ggagcatcatccctcccccg cacccccacc cgtcacgcgc acacatgcgc ggaatcgtcc 1740 actcggcccacaaggggcct tgcatccgat ggcaagcaaa aactacccag tccgtccgta 1800 ggcggggggtcgccagccct gtgggtgggc gcttcccccc ggcccgcaag cgggcccgga 1860 atgggcattttttgcctgcc ctaagatcat aagaagggca aaaaaaacat cgtttcaaaa 1920 cagcgtgttaccacccccct ataggacacc agagtccggg gtagaggact ctggtgtcct 1980 cttaggccatttatgtccaa gaatgtgaca gccagccgag cggaggtaga ggactctggt 2040 gtcctatgcttaggccattt atgtccaaaa acttgacaag ggccacattc ctgccaaatc 2100 tgtccagaatttggaaaaat tcgccggata gtagacagtg gcaaagcctc cccccattcc 2160 cgcaaagcgcccgctcggca cttgggttca aactgaccgg gaagcccacg aggcgtgggc 2220 gatactggcaaaaaagcctg ctgccagcgc tgtgatgcac attctgtgcg ccaacctcgg 2280 tgagcataatgccgtggtca tcagccagga caccatcgcc aagctgtgcg gcctttccac 2340 acggtccgtcaggcgcgcca tcgtcgatct ggccgaaggc cgatggatcg aggttcgcca 2400 acttggcgcgaccagccaga ccaatgccta tgtcgtcaac gaccgggtgg catggcaggg 2460 atcacgggacggactgcgct acagcctgtt tagtgcggct atagtcgtgt ccgaggagga 2520 gcaacccgaccgcgctgaac tcgaccagca agcccccctg cgacacctgc cacgcatcag 2580 cgaggggcagatacccaccg gccccggcct gccgccacct tcgcaaccgt ttctaaaaga 2640 catggagccagacttgccca ccattgaccg ggcaacatca cccaactttg accagcagga 2700 acaggggtgaaaaaggtgga caaactttcc atacgcgagg cggtaaaaca cttcgatgtt 2760 tcccggccaaccctgcaaaa agcccttaaa tctggcaaga tttcaggtgt tcaggatgga 2820 caaggaacgtggacaataga cccctcagag atggcaagag tttaccagcc aaggcaagat 2880 gaggtggtaaaggatggtgg ccaagaacat gaaaatttgt ccgccaagaa caccccttta 2940 catggtcaagttgaggttct gaaagagcgg cttgcagatg ctgaaaaacg ggtggcgata 3000 gccgaggcactggccgaaga acgtggaaaa cacatcgagg atctacgccg gatgctgcct 3060 gcaccggaagccggtcagcc ccgccgccgc tggtggccat ggtaaggtca gctatgcggg 3120 accaagccgcagctcgcaag tgcggcaaca gaatcagacc cgcttcggac agagagctca 3180 agctggtggaaaaccgcctg tgaagctgct gggtggcagc cgctttactc agcgaagcag 3240 ccattcgggcggatcgcagc gagattgccc catccagtcc taagggccgt gtaaacagca 3300 cttagtgcagcagcatcgca ggcgcggaaa tctgcctgcc cttcctcgcc acctctgagg 3360 cgtcagcgatctgccccaaa cctgcctgcc actttggcgc agcatgttgc ttgagaacac 3420 ggagctgccgatctctgcgc taagcagaat ttcctgataa tacgcacaga tgcaaataat 3480 attgcggcatcaatgcgttt ccgtttaccc ctaggcagtt gcctcatcat gctgtcgttc 3540 gctgtccaagccgcaacgac ctgcgaccga accttcactg catcttactt tcggcccctg 3600 cgttcgcccgtgaccgcagc gcggcccaaa gcgcgaccgc cgccaaggcg ggtaggaaca 3660 gccccattgccatcgggaac gggctgtcgg tcccggccaa ggccacgcag gtcgaaatcg 3720 tgaaggccatcccgaactgc accgcaccca agagtgccga tccagttccg gcggcctcgg 3780 tcgcggcggccatggcaagc gaggtcgcat tggccgaaag cagcccgacc atgccgatcg 3840 cgatccagagcggaatgata aacatccata ccgaacccgt ggcagaggcc aggacggctg 3900 ccagcgccgcaagaccatag aacggcagtc cccggttcag catgtcccgg ggcgtaaagc 3960 ggtccaggaggcggctgttg atctgcgcga agacaaacag cgcggctgcg atgagcgcaa 4020 atatcaacccgtagttgagt gcgctcatac cgaagaagcc ctggaacacc ccggacgatc 4080 cggtgatgaaggcgaacatc ccgccctgga ccaaccctgc caccagcacg ggggcaaggt 4140 aggcgggctgtctgacaagc cgcaggccgt tccttgtcgc gcttcggaag ggctgcgcga 4200 cacggcgctctggcgacagc gtctcgggca ccaccagttt cgacaggatg agggcaggca 4260 gaccgaccagcaccattgtc acgaagatcg accgccagcc gaaggcctcg agcagcaggc 4320 tgccaagtgtcggcgcgatg accggaccta tggtcatcag catcaccagc aaggtcatca 4380 ccttggccgccttttgcccc gagtagagat cggcgacgat ggccctgctg acgaccatcc 4440 ccacgcaggccccgatcgcc tgcaaaaagc gcagcgcgtt gaagacaacg atattgtcga 4500 ccagcggcagcgcaattgat gtgacggtga agatgaagac gccgatcaga agcggccgct 4560 tgcgcccgtatccatcggtc agcggcccga cgatcagctg cccgaggcaa aggcccagaa 4620 agaacagcgacagtgaaagc tcggtcgccg catggctggt attcagatcc tcggcgagaa 4680 tgccgatggccgagagatac atgtcggtcg ccagtggcgg aaagatcgac aaaagggcga 4740 gcacggcgatcaaggcaccg gctcgggcag ggatgagggg gcggtccagc atgatatcct 4800 cttggcgggatggtgcgatt ccatttgtag atcgcagtct actaatgtaa aattatactc 4860 aagtctataattcaagtcgc ttggccggaa tcgcaggaga agacaggaat gcaggaggta 4920 cggagcggaccgggacggcc aaaagacccc gtggttgccg aagcgatccg caaggcggcc 4980 ctgcggctggtgcgcgaaag gggataccgg aacgtaagca tcggcgcgat cgcgcaagcc 5040 gctggagtggcgcggcaaac gctttacaat cgctggcacg cgaaggccga cctcatcctt 5100 gatgccgttttcgaagagac cgggcggcgc gccgacgatc aattaccgct ggaaacaggg 5160 gacgcgtcccgtgatcggct ggaacggctt cttataggtg tgttcaatca cctccgggcg 5220 gatggcgatacactgcgcgc attgatcgcc gccgcgcagg aggacagcga gtttcgtgag 5280 gccttccgggaacggttcgt cgcaccgcgg gaaaccatcg tcaccgacat tctggccgaa 5340 gccctgcgccgtggcgagct ttcccgggaa gccgacccgg ataccttgtc gaccatgatc 5400 cacggcgcattctggtatcg cctcctgaac ggacgcgagc tcgatcatga actcgcccga 5460 tcaatagcgcggagcgtgtt tccctgagcc aagacgaaac ggaaacggac ccaaatcatc 5520 ggaatgtataaagtcgcgca tcgttaccat cctgaggggg tgatcgctgc ggagcgagac 5580 gaatggcagctaccgggaat gcggccacgc cgcattggtc gaggtcggcc agggcaaagg 5640 gctggccttcacctatacaa gcgacaggcc tgccatgcgg cgccctgccc aagtgtcagc 5700 gatgggctcaaaccgtcgat aggccagccc cggcccgctc gttccatcgc gggctgtagg 5760 gtcggggctggccggatcag tgagtaagcc cgccatcccg gtcgtggtca cgggcttctc 5820 gctcccgcaaccgttcctgc tgccgctctt gctccttcag gcgttcggcc tcctgcaccc 5880 gctggcgttcctcggcctcc cggctttcct gcaaccgcgc tgccgcctcg gccagcgtgc 5940 cccggtcgatcccctgcgcc gcctcccgta gccgctcggc caggctgcgg gaggattccg 6000 gttccggggtttgtccaggt gcagctatgg catcggacgc ggtgcggctc tggcgggcct 6060 cccatgcctcgcgcaaccgc gcggccaggt cctgcctccc ctcccggcca tcccgatccg 6120 cagcgccggcataagccaga ccctgcgccg ccccctcttg cgtcaacggc ccgaggcgat 6180 ccacagctcgcccgatccag tcgcgcacct gaccggccag aacctccgcc acacgcgcaa 6240 cctcggccgcctgcgccttg acctcgcgcc acacccggac ggccccgacc tcgataccgc 6300 gttccttcatctgccacgcc ccggcagaca gttgcggcaa cggatcccgg tccagttcca 6360 ccgcccgcaccgtttgacgc agcgcctcgg cctcgtcgcc gcgatcatgc gccgcgctgg 6420 cccgctcctgcgcctcgatc cgctgcgcct ctagcgtccg gtggtcgatc cgttcctcat 6480 ggccgcatcgctcaagggcg cggttgctgt cccgcgccca tgcctcacgc catccttcca 6540 gcatctcgaccgcgttccag tcccggttct tcgccccgaa cccctcgggg ccgatttcgc 6600 gggtggtcagcaggatatgg gcatggtggt tgcgatcatc gccggtccgt cccggcgcat 6660 gaagggcaatgtcggccacc atgccacggg ccacgaactc gcgctggcag aattcgcgca 6720 ccagctccacgcgctgtccg tggtccagct cggcgggcag agccacccgg atttcgcggg 6780 cgacctgcgaattcttccgg gtctctgccg cctcgaccgc gttccacagc gcctcccggt 6840 cctgcacccatgcgggggcg ttggcagggg cgagggtttc gacgtgatcg acaccgccac 6900 gcgcacggtaatcgaaggtc agcccggtgc ggtgatcctc gatccgttcg cccacgcggt 6960 aggccgcagccgccgtggcg ctacgaccgg aagagcgaga tatcatcgtg gcccgaaggt 7020 gatagatcg7029 2 4005 DNA Ketogulonigenium misc_feature pADMX6L2 2 gcgtctgtgcgcatactatc ctccgtgttc atcaggctca cgcctgatct gattagggct 60 cttgtctctgcttgtatcgt cgccaaacta tactttaagc agcgtctaga gcctgatgaa 120 cgatctaagaagcccgcccc ctgaaaggcg ggcttttttg atctgtgcca gatgttgtta 180 catcggcgctcaaaaatcaa gttttttctt gactttcaat aatttgcatt atgcacatta 240 ttatcgtttgataataagag ccagaacagc acagaatagt tgtgcgatag ctatgaataa 300 tagcagatccatccctgttt cctttcttac taaatacaat gcgaaactgc tcgcatctgt 360 tttatttagttgaatcggaa actccaaatc ggccggattc aaaaaaaata tagactatct 420 ttaaagtagcaacgccgccg ctcgcgcgac ggcattgcgg gaaatgcgat agcaacaaaa 480 ccacttattgtcgtatatcc ccactttcaa gcgaagcgcc ccgcgaagcg atcaaaaaag 540 aaagaaagaagggggcgctg cccccgtcat gcgcaagcgc atgactcccc cgcccctcac 600 tcaccaaagcagcagctacg aagcaggacg ggatcggcgt ttgtgcatca cgtgctcgca 660 tcaccaacacaaatagacgt atccccatga atggggcccc acgtcgattt gtgttggcgc 720 tactgcgcgcgcaatgccct gcagcctcac ccgaccggct ccgaagcagc agcttttaag 780 acagtgagtgacacaagaaa acatgcgcaa gcgcatgtgt gcatcgcgtg cgcgcgatgc 840 acgggaaggttgccttcgtt gtatataata atatagcctg cacgctgtgc gcacagcgtg 900 catgagaggcaaaaacatgt caaaaatgtt gactactgca caagttgcac agcgctttgg 960 actgtcacgatcgacagtta gcagagcgct taaaaacggc gacttgcgtg gcatccgcga 1020 caatagaggcgtgtggaaaa ttgccgaaga tgatgcgcat aaatggcgca gcgacgccgt 1080 gcatgaacagcgtgcgcaca gcgtgcatga cagtgcatta cgcacacgtg ctgaagttgc 1140 ggaagctcgtgcaacagcgc tagaaatgca cgtgtctgac ttgcaaagcg agcgtgacga 1200 cttgcgaaagcagcgcgacg agctgcaagc gaagttagac ggtcggcccg tcgaaactgt 1260 cagtatcagccagcttttcg ggcgcctttt tcggcgctga catggcgcgt agaagcgcga 1320 cagtctcagctttcgttcgc ttcatctgcg gcatgatctt cacagcatca gtgcgtgctg 1380 cgcgtgtctcttcgacctct tttctagagc cgagccgcaa tgcaatctga atagatctgc 1440 gtattcgcggcaagctttta cgcacttctg aaatcgcttc atcgagcgac ttcaagcgct 1500 ctacaagaatgccgcgcaag ccagaaactc gcttgcgctc agccctgacg gcttcgacag 1560 cgacagacttttgacgctca aaatcgactt gtgcagcatc gagctttgca cgctctgcat 1620 cgagctgctcgcgttgtgtt ttaagctctg tgcgctctgt ctcgacagct ttaaaagcct 1680 cagtttctaagcgatccggc gcgccgtccg acttctcacg cttaggctct agcttgatgt 1740 tattccgacgctcgaaaaac gcagcaaact cgctttgcag cgcaataccg accgcgcgcg 1800 gcgcgctatagtttggcgct gccccagaat gccgccgctg gatctcttcg cgatgtttat 1860 cggccagctcgcgaccaaat tttgtggcgc tcgaccacat ttcgccaggc tgatccggcg 1920 gcgttcttttcgtgcgcttt tcataaactg gcgaagcaaa aacatcgacg atgctctcgc 1980 ccgcctcgtctctgtctagc ctcgctgcaa agacagcgtt accgccgtgc gtttcgttta 2040 taaactccactgcttggcgc accatggcgc gctgcagctc ttctttgctt ctgttggttc 2100 ttctctcgtcaagaagctca ggcggaaacc gcactataaa gtgcagaaca ggcttcttcg 2160 ccgctttgttttgcctaacg ccttttgtgt gcgcgtcata tgccgaccga aggtctagcg 2220 tcttataaacgagcggagag gcatctctta cgacgcgttt cgcacttgtt ttgtcttgtc 2280 gtttcgcgtgcttttcggct gctgacaatc cagccatatc taacgcacta catctcaccg 2340 ctgctttcattttcaatccc tcatatatta ccttttctgt tgtttttgcg aaaaacgcaa 2400 aactcgctttgcgttttttg ttcggcacct gcggcacctc caaaaaacac gcttgctctc 2460 cgagggtctggcaggagcct aagagggggc attctgcccc tgatcgaccc cattgaggct 2520 cgatctaaacagacccccca caggggcgtc tgtgggccgc tagtgcggcc ttccgccatc 2580 ttcgagccatctgatctctg caattagaga tgcatggcgc atctcccatt ccagctcaga 2640 tatcccgtatctgtcgatga cggtgtcata caattcgtcg agccttgctt cgcgctcttc 2700 ttttgtcatgccagttgcat cgcgttcttc catctcctcg ttctcctcta tgatgaaatc 2760 attttcgtcctcgtcgtttg gcccgctttc tgctgccttg accgcaacag cgcctccgcg 2820 ctctgcggcctcgagcaagt tcacgcgttt agcgcgcact tgcttccacc cttcattgtc 2880 ccactccgcgacaatttcag ggccgctttg ctctgctgca tcaacttcag ccatagcttc 2940 atcgtcatccagctcgacca aaccgcattc ttctttcaag ccttggctcc acaccaattg 3000 ccgcctacgcttgccgctcg ttgcattgaa atattcgagc caaagcccgt catcgcccgc 3060 ctgaagtagctgccttggcg tgcgtccttt gcgttttccg ctcttcgagc ttgaaagcgt 3120 caactcttcggcagcgcccc acttcgctac gtagtcgccc gcattggcag ccccgcgaac 3180 gtcaaacgccgcatcgttgc cccacatgcc ataccccttc agacatgcac gccacgcatc 3240 gcctagacgttgcatcagat gcagcgcttc gctttcatcg ccagctctta gcaagacaat 3300 ttcgtgaaagtgcgggtgcc acccatttgc atagctatga gtaatttcag ttgatgtgac 3360 tgacccaacaaatggtaaat cgcgccactc gcggcgctga cgcaaccgct gtttcgcctt 3420 cttcatgttttggagaagat caaaaagcga atcacctgct ttgtgctggg ctgtcagagt 3480 tatgagcaccggcacaaacc cgttgtcgcg cgcccacgcg agcaagtgat tcatttcaga 3540 acggcgaatttgcgcgatgc gagcgctaca aactgcgcag ccccacacat tccggcactg 3600 tgctagacctgaaaagaatg cccgacgccc gccatcctcg cccacgtttt gcacgtttag 3660 ctcaactgtcggagacactt ttacgtgccg acatttcgca acttggtggg gcttgttttt 3720 gttcagattcaacagaatcc gagcggctga acgcagatct gcataaagct gccgcctact 3780 gaataacctgttgttttctt tgtttttttc attcggttga cccccattct ggtcaaccga 3840 tttacggtatataccaaggg gggtctgccg accccctgaa aaagcgtcat cgccgcgcgc 3900 ctgcgcacccgcgttcttct tcgatttctg aactgaatgt gatgctagtt tgtgagacat 3960 ggccgcaaaccccgacgggt gcggcgacta ttctcttgat tttct 4005 3 19695 DNA Ketogulonigeniummisc_feature pADMX6L3 3 cactttcgcc acaagatcag gcgcatgatc tttctcagcgttcaacacac atttgagctc 60 ctgcgcctga tcgtcagtaa ggtaatatcg ttcttgcatcaatttctcct tagttggatt 120 ggttgcactg gatctttgcc ctgacagcct catgcctcgaggcctcggcg atgtcgtggt 180 gcattgtatt ctcgcggcac tgcatcgact tcttctcggtttccttttgt tcgcactggg 240 tacatggatg tcgttgctgg tcacacacac cagagcacacctacagctct cccatgcgac 300 cgccttggcc gcaccgagat acatcgcctt gtgtggttcgcgacttcggc agcaatcgcc 360 gtggcagctg gattggagca tcatcaggca agggcttcgctgacagcgtt cgtcgtgaac 420 gcctaggggc tgtaaaccta aaactgcccc cattgaccgccgccagaaga atgatccatg 480 tctagggtgc cccatgaatt acccgcgata tccggtgtgccgtcgcctgc aacagtaggc 540 agaccgctag caggattgat cgagtgtatg ccaccaaatgtgccccctga aatatccagg 600 gtgctcaatt catccattgt atgcgctcca gaggttgggattgtgctaaa ggattgaaca 660 gtatggtctg gagtacccca ccagttcaga ttcaaaaaacttcctatcat tttgaacatg 720 attggcttcc ttttcgatta agttttcaac aaacagaattaatctacggg ccagttgtgt 780 gcagaagagc gacggcaaat tgctgttata gtatttacgaaaatggtcgc caaaaaatat 840 ctgtcgatca agtgttttct gtaaccatct gaactttttgataaatatca aggtgtgagt 900 ggtttatgcg atgctttaca tctgacatct ggtaaccgcccctgttttct cagggtgtag 960 aatgcagatg acactgaaaa tacaatgaag tgaatgagagccgttgccct gcccagccaa 1020 ggcgaagatt tcattatccg ctgagcaaat cttgtaccaaagagggcttg cgttcaatga 1080 gggccagtag gacttgagct ggcccttgag gcatacggcgcttttgctcc cagttgagaa 1140 gagtgccctt cgccacgccg atactacgtg caaactccgtttgagatagg ccggtctggg 1200 cgcgaatgcg cgcgacatct atctcaggca agctgatctcatgcacagcg gcaccggcct 1260 tttgcccagt cgaaaaggcg cgagcttctt ccaagccctgcatgatactg tcgaacgcgc 1320 tcattgtttg ttgctccaaa tggcgatgat ctctttgctcatttcgaccg cggccgcctg 1380 ttctgtcggg gttaggttcg ccttttcgtt cttggcgaaaacggtgatca gaaatatcgg 1440 catgtggcgg ccgccaaaca cgtagatcgt tctgaaccccccactcttgc cggcccctgc 1500 cctaggaatg cggacctttc tcaatccacc acccaaagcgatgccggcct ctggattttc 1560 tgcaatccaa gcaattgcag cgtcacgttc cgcatcggtcatgatcgatc gtgagcggcg 1620 ctggaactct ggcagttcaa ctacggtctg caggcttgtcatttaggtat acttcaatgg 1680 cgcataagtc aatggcgcac tacggcttct tgagctgctcaaggagcgca acagcgtgtt 1740 cgagggtttc accgaggccc caaccgttgg catctgctattctgtaaaaa gattctatcg 1800 tttcaggcgt cgccttaatg ttgaactgcg cgtttcggccagtcctacgg cgccgctgcg 1860 gcttggaaac agggggcgat ttaggctccc tgctcacgaagccagacgcc tctgccgctt 1920 gttcagtgct gcggtcttta ggtgctatct tcggagcaggcgcgaagctg ctaagatcgt 1980 ttagtgcatc accaaaacca aggtttgcgc gttgcttgctcatcactgat cctcttggct 2040 acgcttcagc tttccaacca cttcgcccgc aaattgccgcgcgttctcga ttgccttatc 2100 gacgttgctg acctgcgacg gatcaaggtc agacaaggttccgccatagt cgaacaagtc 2160 acgataggcc gcacgctcga caatcgatgt ttcgcatatatcgatgccac cgctcatgag 2220 ctgctcgtga acgtttttca gggcgcgtga gcgaaccgcggcacttgttc gggtcagcac 2280 aacgcagtgc ggtatggcac gccgcgccat cttcgcttggttgctgatca atcgaatggt 2340 ttttgcgcca cctctcgcat ccatagacga gccctgtatcgggatcagta ccaggtctga 2400 cataccgata gcgtttgcga ccatcaggct cgcagtcccctctaagtcga cgatcacgaa 2460 ttgagacgtg ccggatgccg cctcaatctg gtcaacaatgccgtcttctg tcacgccgct 2520 tacgatggaa atattttcag gctttccggg taggctcgcccattgggaga tccagcgttc 2580 tggatcggcg tcgatgattg tgacgcttgc cccgccctccgaaagctgcg tggccaagat 2640 caaggctgat gttgtcttcc cagccccacc ctttggattggcgaatgata tgacaggcat 2700 gtgtgatcgt ctccgcgctt ttaggtacaa tatcagatagctaacagata tccatatata 2760 agttacagta tctagtagct atcggatatc atacaatactaaaatggtac tatttggtat 2820 catatgccaa atatcttgac ttcatgacca cttggttctgcggcaggtcc gacgaaagag 2880 cccggcgagg acgcttactt tttctttggc tgccatttttttgcgagctt taaaacgtta 2940 cgacggagct catcgccatc ggggatatgt gggtcgaatggaccttctac ccattgggtg 3000 agttcctcgt gttcagggtg tgcggcgtcg ccgattgcctcgatgaagtt ttcatatccg 3060 ggcaggccgc cgacgtcctc aggcggacat ctaccgacaacatcgacaag ccgtgggtag 3120 aggttttcgg gaatcggatc gtcgacgctc tccgtctcaatcagatgaac ccagtaatct 3180 ccgaaatcgt aaacatatcg gatcgggtcc gctacggcagtgttgatgat gtcagctagg 3240 atgtccgctt cggaggtatc ctcgtagacg cggatcagtgcgaaattggc cttgagcgcg 3300 gccttgtcct tcaacttgtc ccgttcaatc actttgcgcgcactggcgct gtttttgccg 3360 tggtggcccg gcccgtcata ttcgatgatg gcgcgcacaaagccgtcctt gctccaaatc 3420 acgaaatccg cgcgctttgc gacaaggccc cgccacagattgccttgagc ttggatgaag 3480 gcaccgtatg gcacctgcgg cgagagaata agatcaggtctgttctcgtg ccgccagcgg 3540 ttaagcaggt tatagagccg aaattcactc ttattcatcaattgcttgct gctgtagagc 3600 gattttcgcc cttcaacgaa ccagtagaca cccccgacaagggcgatgac caccagaaat 3660 ccaattatca ttgtgaaatt aatgtcggtc atgactgcactccctcaagg ccgaagtcag 3720 agattgcatc ttgacgcaga gcgacgagtt caacgccgtcgatgcggttt tgggtggcgg 3780 tagccgggct ggcgcggata cgcctaccgt tggcacggtttctaggcgac atactgaggc 3840 aaaccggatt tccagtgggc tgcgtgcttt gttgtaagcgcgccatttca tccccccgag 3900 ggcaggggaa cggctcacaa cttccaaagg ccccgaaaaattctcgcgat gaccccacca 3960 cgggatttgt gcaacacaat cggcgcgcgg gacaggctcatatcaacttg gataagcgcc 4020 gcgttttttg caaaatgcga gaaagtgctt atccgcatctttcacgttga tggccttgtc 4080 atgcacccaa gaacgccact cttcttcaag ggagtaaacatcccacccag gagcaagttc 4140 acgggcagta tcgcgtgtgt cgggatcgtt gaacggcaaggcctgaaaag tggttgatgc 4200 tatagtttcc accaccttgg gtcggaagac agcgttctctccttcgatac tcatgctgta 4260 gtcagggaag tggtcgtgcg cggtgtcatc ctcaatgatcttcgaaagca ggcgacggaa 4320 ctctttttta gttgagccgg aaccgcactt gtttcgcaagagctccaagc tacacatcca 4380 ttttgactgc gcgccacaat gctttcgccc tatctcatacaaccgccttt cgaggggctt 4440 tctgagcagg aaataccccc ggcttagagt gaggacatggttgttctcga tcgcatcaaa 4500 gacccagtca gagagcgtta tttcgacatc aagcattcggccgtcgcggg ttacgcgcac 4560 gatctcggct gattcaatca ggccaaatac cttgaagtattctttcccac cttgacgaat 4620 attcgtttca atttgggttc cctgcagccg ccgaagggcatctttgagca gctgataacc 4680 ctgaccggat gtctgacggt tcgttgccac aagcagatcataagccttga aacgcatcga 4740 tctacttatt ttctgcccct cattaatggc cgccatgcactggctgatgc agtagatcag 4800 cacatcacgg tcatgaacgg tagcaaggcc atagcgtgaaggggaaacct cgatccaatt 4860 gtcgttgttc tgatagcggc gtggcttcat gtccggctttgtagacaggg tgaacatagg 4920 gtgctccatc gaagccatat cccccttggg aaccgcatcaacgatgtcgc aaacgaaaag 4980 gtctttttgt ggatgacgat ccggcaaaag cggtgatcgtaggttcgtca tttcacacac 5040 tcccgcgcca agtaaaaatt cgtcatttca cacaccgtacaagactatcg tcatttcaca 5100 caccactcgt caaggctcgt catttcacac acccaaggaggctgtggata actcgagccg 5160 ctatcgtcat ttcacacacc atctttcgtc atttcacacaccatctttcg tcatttcaca 5220 caccaggtgt tatttttttt atagttatat caattgattacgagctgttt tcggagctgt 5280 aactctattc taactctatt ctaactctca tagcttgccaaaatggcacc tcatatcccg 5340 gatatccggt ttcattatga aaccaatcaa caacatttacggtgtttttt gaggagcaac 5400 actgtcccaa cgcaggttca aaccgatcac gccgaatctgcaaagaaagg ggcagtgcta 5460 tgttcatttg gtcactcgag gatcacccga accacaggtaagccctcaca tgctatgttg 5520 atacctccag ggaagtagca aaggtcctca cgaccaccacctgcaggatc tccatcagcg 5580 aagacaactg tcgcattgaa cgctggcgcc ggcttgtacgtactcggtat gggtgagaag 5640 tcaggcatac ctgggagaac acgacgaagg acaccgtggttctcgaggat cccgtagaag 5700 atatcgggcg tcccattatc cacataagcc gcaccatagcgatcctcccc gtagagatac 5760 tcacggaaag tttgcatatg ccgccagcga gagcgaaaaaggggaacccg aatagcgaga 5820 tcctgatcgc ccaaccaaat gtcagccatg atgaacatggaatattgatt gttccacgac 5880 gacaaagccg caaattccat gaaccgtctg accgcatgcaggtcagagcg ctttggctcc 5940 atacgatgaa gagggtttac atctcgttca acactgcgacgatccgcctt cttcaccgcg 6000 ctcacaaatg gactgcgatg taatcggccc tcaaggtagccttcaagctg ttccgcatag 6060 gcctcatcac gcttagagac actctccaag aggcagtcagaaacgacctg acgccagtac 6120 ccattcccac gcgctgtgtc gtgacctatg acgggcctctgattgtatcg cagggagagg 6180 tgcgtaaccg gagactcgaa gctctgagac cattcccagaggctctgagc catgggatcc 6240 cacgaaggaa ggtccgtctc tggttgctct atctcgccaaaacctaaatc ctgcagctgc 6300 acattcgcag gatagacttt gaacatcgtt cgcaccttgccgaggtcatg aacaaagccc 6360 gcgataaaag ccgcaagatc gcccgcctct gaggggacggccttactgaa ctcactgttg 6420 aagaaatcta ccgcaagccc agctgtttcc agggagtgatagaatagccc accggctgtg 6480 cggtggtgat gtgtagatga agctgggagg cacagcatatagtcgtggcc tccgcgcaac 6540 atttgcctca ccaagagtgt ttctttgtga gagaggaaggggagtttttt gagctgctcc 6600 ttgatcagct gctcatagct tccaaacgtc acatgcgggtgcaatatccg gaactgagcg 6660 ttgcgcaggt agcgcccgta agacaggatg atgcgatcgtcataaaacat tggcggtttc 6720 ctcaatcgat atttgctctt agttggtagg tgcccgtatgggcgcggcac aggtttctca 6780 gctgtgccag gcggtcagtg cgatagcgga tccaggcgaagttggcctcg cacagaagca 6840 gttctctacg gatggcgtct ggaaagcgcg cgaagatccgatcgctgtat ttgtactttc 6900 ctgacatgcg gatctgttcg gttggggtga acttgtcacctcccatccgg tagcgggcct 6960 tgccttggaa tttgacccag taaatgcgcg ggcccaatgtgtcctcgcgg attcgaacga 7020 caatgcccaa attcagcgcc tcggcgcccg cgtccattttgcgataggcc ttcagggctt 7080 cgacagcccg atcctgccaa tagcgcgccc atccttccaagtcataaccc atcgctggaa 7140 tggatcgcaa aagctcaggc tcccaatcgg agtcgaaatcatgaaagcgc acttcgtgcg 7200 aacagctctc ttgggggtgt tcttcagaca tgctgtgctccgaccaaatg gcgcgccgac 7260 caaaacaaga aggttgggtt caatgccggg atgtttctgtctgcgttcag ataaagccca 7320 tgtttcctaa tgtgacggga tggtagctcc gtcacattagggggcgacgc attttgcgcg 7380 gcacatggcg cggagagccc gcaggcaggc ggctggacggacgaaaatgg gcaggggccg 7440 cgctgagatc ccttgaaagc ccatgggcct aatgaatggcgctcacacag cctgtcatgc 7500 gtgtacttta cggggaggtc tcgcgatgag gtgagcgcccgcggtctctc gtcttggtcg 7560 tgtctgaccg tatgtgtccg tcgaataaac gtcatgatgaggtcctgaga taagaacgaa 7620 caggatcgtt gacggagggc atccgtctga ctgtcgtctgatgttgatgg aaagtcgccg 7680 gctctaggcg gccggtctca atttggtggt gagcagctctttgcgctggg gggcattacg 7740 atgcagatca gccgtccagt ttaaatcggg aggggtgagggaggtcgggg cggccgtgca 7800 caagacaatg gcatccgaaa gccggtcaat cattgggcgtatttgtccta cccatctgtg 7860 ccaattcata gcaaaaatcc cgaaccatcc gtttcgtcacacctattggc tagggggagg 7920 tggggatggc cgtcaatgaa aaatatcagg gattgagttttataattaac tgaaattgct 7980 aaatattttt ggaaggaata ttccttccgt agctcgtctactcatttatc ccttttcgaa 8040 aaaagactta acagcgggcc aactgtttcg catgatgcggacaagaaaaa ttggctatca 8100 tgggagtgtt cgggtggcgc gtgggactaa gacagccaaaagcaaaaatg ctgatccaat 8160 tgtagcagaa tgcttaaaac tgattcgcga agacagtgggtatgagagag atgaattcgc 8220 cgaacttcta ggggtgcagc ataaaacgta tcgcaactacgaaggctgta tatatccgct 8280 accgttaaaa gtggtaaaga cgatacgtga gaagttgggctatgatcttg cggatcctga 8340 tctgacttca gacgcgatca tcaccaaaat tgcagagcagcgacacgatg ttgcagccgc 8400 tcctgatctt gccgcgacag agcaagtagc gaagggcgttagttgccctc agaggatacg 8460 tacctgcctt caagctttcc ggcaggaact cattggtgtccagagcaagc gcaagcatga 8520 tattcgagac gcagtttttg tcggagcagc tgcattgtttgctttctgct tggtggtact 8580 acgcactgaa ccgcaaaata taaggttaga gtctatctatacccttatgc tatccgtgtc 8640 cttcttggtt gcggcgtcaa ttgtcccgtt tcaggctatacatatgatcc aagccgccta 8700 tcggtcgcga cgttgaccaa caatactagc cctccccgtatgcccgtctc acagggaggg 8760 ccaaagctac cttggcgaac gactgtcagt gcacgaaacgggtgattaaa cgcaaagtgc 8820 tgactttttt tcccccgata ccgatggttc atcaaagatatatcgactga gcagatcggt 8880 gtccggggca cgcgtgtgca ttgcttctag gccagaaactgtgaagttct cttccacgtc 8940 ggattccggc acaaggaggt aatgccaggg cttctcaccgctctttgcac cggcttgcgt 9000 cgcaatatgg caccacaggc ttgccgcatc tgctttacgcagaacctcgg tggcggtcat 9060 ttccgattgg cgttttacct cgatgatcag cttgcctgtatcggtttcga caacgaaatc 9120 gggctgatac ggcgcaccgt gattatcgaa aattttgaactggttggggc caggtttcat 9180 ccaaaggcgg accgagctgt ctttttcaag gatgatcgccagcttgcgtt ctggattaga 9240 gtcgaacttc gcatactgat agcagccctt cacgaagccagtgaagatat acttccggat 9300 ctcctgcttt tgatctggcg gtgttttgaa atcccggatgaagctcgtac cggcggtatc 9360 gaaggtctgc ggacgcagct cgccgaatgc agaggtcagagaaacacggt agttcgtgtg 9420 ctcacgccac atattctgct ttatttgcga gaaaatgctttcggccatcg ccttcgcatg 9480 cccccggacg acactgcgag tttgctcatc gtcgtccgcgaagcggttgc gggtttgggt 9540 gactgcctgc ccagccagat catacaagat ggcagcatgggcgtcatagt cgatctcagg 9600 gtagtcgatc agccgcgcga cgatgtaatt ctccagccggtttgcggtgt cccccttatc 9660 ctcgctggaa atacgcgatg tcttctctgt acgcagagcttggatcatga gctcattgga 9720 caacggctgg aagttccagc tcttcatgtc caggtcgaagcgcttgaagc cgaaggaaac 9780 ttgttgctgc ggagtgatcg tgagagcagg gatcgcaagggtccgctcca cgaaattctt 9840 gcacagctcc tgagcgaccg cgacggcctt ttccttcgtgatgctgggaa ggaaaccttc 9900 ttcgggtttc tgtgctgcaa tggctgcttc cgcgatgcgttcgatcacct tggggtcgtt 9960 gagatcgcgg atcgaggaca cttccttgct gagctgggggatcacaacac tgagcaccgt 10020 gcgggcgacc ttcagctctt caggcgtgct gaagctgaaaggcgcttggg tcggtgccgc 10080 agacgtcggc ttggcgacta gctgcacagg cgcgtccgaagctttttcgt catccgcgac 10140 gacgactgtc ggctctgtct tggcctctgc ctgagcaagcatctggtcga ggatggaggg 10200 tgccgagacc gaaacaggct tcgagggcgg cacgtcaccaccctcaccaa tcgtcacctg 10260 cttcagcttg cgcgtgacgc cgttctcttc cttggctttctcgatcagct cgttgaagcg 10320 ttcgtgggca atcacggtca gcgtgtcaac cacttcgacgccggttcgct tgccataggg 10380 caggcgcaga ccgcgaccga gcgtctgttc ggttaggatgtccgaggccg aggcgcgcag 10440 cggaacgatg gtgaacaggt tcgagacgtc ccagccttccttcagcttgt tgacgtggat 10500 gacgatgtcc gtgtcgccgg ccttctcgat attcagcaaacgctgtgcgt tctcgtcgct 10560 ttcctcgccg gtcagcttgg agtggatctc ggccacgcgccccttgtagc gtccgccgaa 10620 gaactcgtcc gactggacga actcattcac ctgccgggcatgggtcgtgt cctgggtaac 10680 aaccagcatg aagggccgca ccaccttcac gtcattctggcgggcatagg tttccagcgc 10740 caccttgacg tgctcgtggt agtggatgcc gtcttccagcttgatccgtt ccagcgtgtc 10800 ttcgtcgacg gtcttcgggt tgaagttcgc gcgggtgccgacagccggtt ccttaacata 10860 gccgtcttcc atcgcgtccg gcagatcgta gcggtagacgacgttcttga agggctgcga 10920 gcgcgcgcca acggtcttcg gggtcgccgt cacctcaaggccgaggatcg gtttcagctc 10980 ggcaatcgcc cgcgccccgg ccgaaccgcg atagcggtgcgcctcgtcca tcagcaggac 11040 caggtcatcc agttcggaga ggtaggagaa gtagctctcgccgatatatt cctgaaggcg 11100 cttgatccgg ggggcgttgc cgccgcgtgc ttcggagttgatcttcgaga cgttgaagat 11160 gttgatgatc gcgccttctt ggccgaagag gtcggtgccacgcacaccgc ggccttcctc 11220 gtagttctcg gcgttgacga tcagcggcgc gttatgggcaaagacttcga tgccacggaa 11280 gacgtatttc gggctcgacg gctggaagtc ggacagcagcttttcataga tcgtcaggtt 11340 cggggccaga acgaagaagt tccggctttt gccgatcatgtagaggtagc tgatgaaggc 11400 gcccatcagg cgcgtcttgc ccacgccggt cgccagcgcaaagcagacgc tcgggaagtc 11460 gcgctcgaac tcctcgaagg tcgcgtcggc caggtcgccatagacctcgc gcacggcggc 11520 gcgggccatg tcgatatcgg cttccttcgc ggggccgacgaggtcgacga tgtcatcgag 11580 ccgccgcagc gcctcggcct gcggcttgcg cagcgacaggcgttggttga tctgaaggac 11640 ggcgcgtttc gggtcggtcg tctgcatggc tcagttctcctctgcgccaa acagatcggg 11700 tgtgtctgcg gctttggcct tggccgattt cttcggcgcgggggtgtctt cgatgtcgtc 11760 cggctcatcc tctgccatcg gcagggcgtt gatcttcaaggaatagtcat cctgccccca 11820 ttcgcagcgg tcaagcacca cgcgcgggat cttcttcagggtcaggttgc tcaggctttc 11880 gccctgggcc tcataggcca tgcagcagat cagcaggctgcggtcctcgc ccacctcgtc 11940 cgagatggcg cggagctgtt cgatggtcag gctggccgttgtcacataga tgaaggcgtt 12000 ctccgaagcc tgaccgtgca tccagtaggc ctcggttgagggcgcgtaga catagttgaa 12060 atgcttgcac atggcctcgg ccagcatctc ggcattgtagtccttggaga tgacccagtt 12120 gccccagaca tccttctgaa gcagagatgg cgcaagacggaagaaacggt agccgccgcc 12180 gcctctccag tttgtggcct cggtcacgcc gcccttatcggtgccgttga tgactttttg 12240 cagccgaggc gcgacatggg ttttggcatg gtcccccagctcgaccatga tccagcggcg 12300 gcccatcttg tgggccactg cacctgtcgt gccagaaccagcgaacgagt ctatgacaag 12360 gtcgcccggt tgagttccta tctgtataat acgctccagcagtttttctg gcttcggtgt 12420 agcgaacgta gcgtctccca aaccaagaag cgtgcgcaggtcattggttg cttcacgggt 12480 tgtaccagct tcttcaccga accaaatgct ctcaggtacgcggccttctt gatcgcagag 12540 atagatcttc cgaagaacac gcgtgtcctc gtttaagaatgtaatttccc cggtctctac 12600 tttgctggca aaggtctcct ttgaccacct ccaccctttctctggcggcg gaatgatctt 12660 cccagaaggg gtagtaacat cgaacatgag attttctctgtagttcggac tacgtacatc 12720 tcctgctcgc caaggtcctt ttggatcttt gtctggattattatagtttt tgttgtgctc 12780 aggctttctg ggcagaaggt tgcgcgagaa cgcctctgttttccgatatg ctagaacgta 12840 attatgatgg agactgacgg tcttggcgtc atttttcccttgaacgctat gctgccagat 12900 gatcgagccg aagaagttcg atcggccgaa aatctcgtcgcacataacct tgaggtaatg 12960 cacttcgttg tcgtcaattg tcatccaaat ggagccgtcttcagaaagta ggttccgaag 13020 aatttctaac cgatctcgca tcattgtcag ccaaagcgagtgctccagcc cgtcatcata 13080 atgctcgaag gccgacccgg tgttataggg cgggtcgatgaagatgcact taaccttgcc 13140 ccgcacggca gggtcggttt ccagcgcctt gagtgccagcaggttgtcgc cgtggatcag 13200 catgttgtcg aagatgtcgc cctcgcgccg cgtgctggcgtgatgcgaga attccggttc 13260 ttcgatcaga atgcggggct caagccgcgg gcggttgttcttgccgatcc aggtcagttc 13320 aagtttggtt ttggcggcca tcaggcaagg gtccatcttacagtgaatag ggtcgtcatt 13380 tcgggggtca ggttcagctg atctgcgatg tcatcaagcatccgctcgcg ctcagcgtcg 13440 atttcgcgca ggcgggtgta aagctggtgc tgaaggtcatccacctgccg ctgcaaggcc 13500 ttggcctcgc gttgcagggt gaccttttct tccagcccgatcgtggcacg ggccagtttc 13560 ttcttctcgg tcgcttcctt gttcagcgcc ttgatctgctggtcgaagga caccttggca 13620 tcctcgcgcc aggcatcgag ccgctcttcc tcctcgttgaggaaggagct gagccgatcc 13680 tgtgccatgc cgatgatcgc ggtctggcgc gcgttcagggtttgggtcag gtcgctttcc 13740 ggcagggtgt ccgcgccaag gccctcggtc gtcgccgggacatagagcat ccgagaggcc 13800 gtttccgggt cgatggctgt tccgccgtcg ctgaaggctgcgaagaccag ctcgtcatag 13860 accttggctg gggttttcag tcggacccgc gccacgcgcatccagcccga ctgccccctg 13920 agctgggcca cgtcgcccat gttgccctga taggcgctatagtcgaggcg cagcatggca 13980 gggaccagat cgcgcgactt ggccttctgg accagctgatcggccagccc ctcgtcgcca 14040 aggcggaaga aacgccagcc gcgctcgtca gcctcgggccattcgctgga ccaggtttcg 14100 cccccgtaat cgaagcgctg cgcatggtcg tcgtggaagcgggcctccgg cagctcggcc 14160 cgcgcgacac caaggagggc gcgcttgaag tcaccgatggcggaatgcac ggcgtctttc 14220 cggccaagaa gccgctcgat caccttgtcg tccatctcggccagcagctg atctcggacg 14280 ttcttcttcg cctcgtcgat ctcgacgctg aactcttcctgaaggcggtc gaaggcggcg 14340 tcgatctggt ccgtggtgcg gcaggactgg acgatgtcgaggatgcgccg ctcgatgtcg 14400 acgccggatt caatgacgcc cagaacctcg tcggaggagccgaacacacc ctcgaaaagc 14460 ttgaacttct gttccagaag ctggtggatg cgcgcttcagcgtggttctt ccggttcagg 14520 aagttgatga cggtcacgtc gatcttctgg ccgtagcgatggcaccggcc aatgcgctgt 14580 tcgacccgct gcgggttcca gggcaggtcg taattgatcagcagggagca gaactggagg 14640 ttgatgccct cggcgccgga ctcggtcgcg atcagaatggtgcgatcgtt gcggaaggca 14700 tcgacgatgg cggccttcat atcggcggtc ttggagcccgagaccacatt ggtgtcgccg 14760 tgcttgtcga gccaagcctt gtagagcgct ttgctgtctgcgtccgagtt ggagccattg 14820 aggacgacgg tctgtccctc gaagccgctt tgttcgagcagatcgcgcag ataggtttgc 14880 gtccgcacgg attccgtgaa gatcacggcc ttgcgctggccgcccttgga gacgatctcg 14940 tcgagcacgt tcggcaggca gtccaggagc gccttgccttttgcgttgtc cgagatggat 15000 gcggccagat cgcggtactg ggtgagccgc ttgatttccgcttcgagctg agcgggatcg 15060 acgcgctcag catcttcggt gtcgtcttcg atcgcgtcggcttccgaacc ggaatccgcc 15120 tcgcgccagt cctcggcttc gtcgctgaaa ccatcaaggtcatcgagcgt gtctgccccg 15180 acaacacgct tcgcctcaag cctgcggatc atcttgtcgagggtctgcat cacggcgaag 15240 gaggaagatc cgaggatctt gcgcagcatg agcgtcacaagatgacgccc gttctgcccc 15300 aaggcgatgg tgctggggtc ttggagatac tcggaaaccttttcgtagag atcggtttcc 15360 agtcgaccag gcgtgaagtc gaaggtcttg gggagacggttggtatagtt gatcaggcca 15420 gcgcgttgga cttggcggcg cagggttcgt ttgcagataggctcaagacg tttcgcgagc 15480 agggcctgag aggtcaggcc ttctcgtccg ccaaactcgcttcggaaggc ttgttctgaa 15540 ccgaagtagg tttcgtcgat gatgctgatg agtccgtacagctccatcag gttgttctga 15600 agcggagtcg ccgtcagaag gagtttctga cgcccagccagtgcctttcg cagaaccgaa 15660 gcccgagagt tttcggcggc cttgtagacg ttacgcagcttatgagcttc gtcgaatacc 15720 acgaggctcc aaggcgtgcg tcgcagagtg tctgcaattcgtgcggcgta ctcgtaggag 15780 acgatgataa tgccttctcc gcgccctacc gggtgcggagtgccttcgtt ctcaaggtct 15840 ttgacgcgct tggcatcaag aatgaacgat ggcagcgagaatttttcacg cagctcggtc 15900 gcccactgct tgcgcagaga agcagggacg atgaggagaatgtttctctc acgttcccac 15960 cagcgctggc taatgaccag cgcagcttcg attgtcttccccaagcctac ttcatcggcc 16020 agaagaacgc ccttcgacaa tggtgagcgt agggcaaatgtcgcagcatc cacttggtga 16080 gggttgaggt ccaccttggc tgccgagagg gactgcgtcagagcgtcttc ttcctgaatg 16140 ccttcggatg tcaggtggtg cgcaaagaac tttgattgatattcagaaaa ctggaagagc 16200 aataggtcag gctccaaggc atattagtta agggaaggttgttctaaatg ctgtcaagtt 16260 atggcctgcg gcttgatcgg ccagcacgaa ggactcccttctgcccagga tattgggcct 16320 tctcgttatc acgccactga aatgtcacct gtactgcgataccatgcttg tcggctattc 16380 tggaaacttc cttccaagcc tcctgggctg attgcccttggatagagatg ccgaggtcag 16440 ggtaatatcg gaatccttcc tcttcgtagc acgccgtcttggtaggaatc tgtatggcct 16500 cagctaggtg atctccggat aggcccttag ccttaagagaactcactgcg ccgagaagaa 16560 ttgctgccca gtttggcttg tcgatcttct tgtgatcaacctccgcagat aagacttttg 16620 taaatgaaag gcctggagtg gatttgaact ccatgaagctgctgatttcc gacgtgggat 16680 caatgctgat ctcgatgtca cgctctaggt caagcgctgccatcttctcc cgtaccagca 16740 gcatgatcgt ttctgatggg gtttctgtcc ccatccaagtcgagatgcac ttcagatcga 16800 caaaagttgg gtcgttcagt ctgactacgg gcataggcgtcatccttcta tattcatata 16860 gaatatgtta catattcaat attatggtgt caatgctagcgtttgattgg caacctgtcc 16920 taaaacggtc gtttttgggc atagtgggat gaagtggtcaaaatataggc cttgataatt 16980 ttgaacatgc ggctactttt ggacagggtt ttttgacacggagggccata cattgacaga 17040 gcaaggccgt gtttttgcct atgtacgtgt ctcgacactcgggcagacag tcgctggaca 17100 gatgcaggaa attgcggcgg ctggctttca gcccccagcgtatcgtgtcg tttcggaaac 17160 catctctggc agtgtcccgg cgatgcggcg accagagttcgcgcgcctag ttgatcgctt 17220 ggaacctggc gacattctag tcgtgtcgaa gcttgaccgtctcggacgcg acgcaatcga 17280 cgttacggag acagtcgctg cgctctcgca gatacctgtccgagttcatt gcctcgcttt 17340 aggtggcacg gatttgacta gctcgtctgg acagttgacgatgaacgtcc tcagcgccgt 17400 tgcgcaattt gagcgcgatt tactccgtga gcggaccagtgcaggccttg cagctgcaaa 17460 agccaaggga aaacgactag gtcgtcctaa agtgctgaccgaggataacg agagcgaagc 17520 acgagcggct ctcgccagag gggaaactgt ttctgggattgcaaagcgtt tcaacgtcag 17580 tcgggcgacg atcggccgcc taagagatag ttaggctatcttgcctatgt cgtcgtgacg 17640 agtcctccaa atacacggat ggcttcacgc tcccatgcacgattaaacca agtaacatgg 17700 cggcttactt ggtccttaat ctcagggcat cccatgatgtccgctgcctt gtaagcatac 17760 cgcaagaaat ttcggcgttt tccaagatgg gtataacgcgccaggctttc tcgcttgaat 17820 acttcaaacg acggtacgtt tttcgccttt gcggcgatcatcttggcatg aatgccccgg 17880 cgcatttttc ctcggtaggc atcgagtgac gaaggtcggatcttcgtttt ctgcccgtcg 17940 aaggtgaaac ccaaatactg gataggtgtg gcagaggccaacagtccgtc cttgaagtcg 18000 gcagtgtctg ttttgtcgat ggacatggaa aggcaaaaatctgccagcat tttctctact 18060 actgcaacca catgatgcac cttcgcgcct agaggcagagtgaccgcgat gtcatcagag 18120 tatcttcggt aagatccccc tgccctagag caccaagcgatcatttcacg gtcaaaagtt 18180 cgaagataga tatttgcata caggccggac accggggtgccctgcgggat accgaacgtc 18240 tggtcatgct tccgaatgag gccatccttt cggcctcggacatgatccga aaagtctgaa 18300 ggtgagcata tccttccgtg cccatttcgc tttcgaccaagaagtttgtc tagatcttca 18360 gtctcaaccc atgagtagcg ggtcacgttt ttccaaacgcttgcatggtg cccttccagt 18420 cttgtttcgc ctatcaaatc ggcgatctca tcgcggagcaaggtgtgatc gagacagtca 18480 aagaatccgg aaatgtccat tgcgaagacc gtgcaatctccacgagactt aatctcgtcg 18540 aacaacgcct ttgcatggtg aatattagtg ccgcccccacggcgataggc tagcacggag 18600 tctgatgtgc cgtcgcgcca tagcgcccgc tcgtacattctattgagatg tcccgcgtag 18660 gcttgcaggt aggctgcatc ctcatgactg gcgaagcggatcggtcgctc tttcactttt 18720 acctcacggg cgccatcctt gttcctgaca taccttctgttgacgtcagt aaacccaagt 18780 aaaggtagaa atctgtgagg cttatcttcg accgagaagtcaaacgacag ctcccgatcg 18840 actaacggaa gatcgaaatg cttgtatttg cgttccttggatatggccgg aagcacaaag 18900 tcgtcggttg atggatcaaa ctcgttttca gacggcaggtaaaacccggg catagcgact 18960 ccgttcgtca aatgcccggg ctattgaacc tagctcccatcaacaggcac cgaggtgccc 19020 tccggtacga gaaaacctgg cgtggtatgt tgtaaccacgatgtgcgaag gatgcgccat 19080 gaccgaaatc acgaccaccc ggtgccgctg ggcgcgggtagcgcttgcgg gccttaacac 19140 tggtcatccg ggtccacgcc ccggggcaaa atatcaattaggtgctacct aattgaatgc 19200 aaggggacgc accaagaata aaaggacttt ttcagctttagtggcgcctt atagtggtca 19260 tctggaacca ggacaaagag acaaccttct aggtaagatcctcgtaagtt cgtatcagtt 19320 acggtgtcag atagtagcag ataacgcacg gcgttctgctggcggagggc gagagaaatc 19380 tggttcgctg tgagcggtat ttcttgccca caacgcggcccttattccag tctagtacag 19440 gcttggcagc caattgccgc gactggcata gcatcgtgctccttgattac atgctaaaga 19500 ggtactttga ggtcttagcg gtcattcgac gattttgctcagataacagc gtaagtaaac 19560 gctgccccaa gtttcaggat ctagcgccca acgatcttatcacaagagac catgttaatg 19620 cgtctattac attaggatgg ttcaaagttg ggttcgggatcatctggtat tggggttcca 19680 aagaccaatt cgtgc 19695 4 4211 DNAKetogulonigenium misc_feature pADMX6L4 4 gcgtggccct tcccgccgagctggacgccg aagcccaagc ccggcttgtc cgcacctggg 60 cacgggacca cctggccgccgccggcatcg tcgcggacat cgccattcac gagccgagcc 120 gcgaaggcga cgaccgcaacacccatgccc acatcatgac caccctgcgg cgtttcgacg 180 gggcaaccgt ggacggatgggcgaagggtg ccgcccgcga cctgaacgac aaggcgttcc 240 tcgagaacct gcgggcatcgtgggagaccg cccagaacgc cgcactggag gccgccgggt 300 caaccgcacg ggttgaccaccgcacccttg ccgcacagca tgaggacgcc ttggccgcag 360 gtgatgactt actggccgccgtcctcaacc gccccccgga acctcacctg ggcgtgtcgg 420 cccaagcgat tgaccgccgggccgggcgtc cggtcagcaa ccggggccgg gccttggcgg 480 aggtcaggga gacccgcacccggctcatga cggcatacga cacggcacgg agggccgccg 540 tgttcatcgc aacgaccaccgccgggcttg ttgaccgggt gtccggtgcc cggtcagaga 600 ccggacaccc cattgcccgaatgttcggcc ttggccgtca tgcggcaact gttgccgaca 660 ccgtgccaga accgtcccgcccatcgccgg acgacgatac cccgtccccg tcctgacccg 720 cctcaaaccg tgccgatgcagcggcctctt taagcccact ctgtgcctct tcggcggccc 780 tgtgctgtct ggccatccagaaatccagaa tggccgcacc acgcatggct tcggcctcgg 840 cgtcccgtgc ctcgtccagacctgcccgga ccaccaccgc ctcagcggtt ccgtgcatgt 900 ccagatgtgc ggccaccatgtccaacacgg cggccctgac ttcctcgggc gacacctcgg 960 cccccatcca atcgtggccgaacttctcgg ccacgatgcg tgacaccgcc gccttgcaga 1020 aggtccgggc atactgctccagaccgtatg cagccttgtc ctcgaccgtc cgagcgtcag 1080 gcgccttgat gaccctctgaactgccattg atgccccttt ctcgacttcg attagtgccg 1140 caatttgctc gaccatgctacgaacccaat ggacggtccc cagcagacgc cacgccccat 1200 tccgtgccag acctgcggccccggccttct tgtccgattt gggcgaaaac cggaactcaa 1260 cccgcacaag atgcgggtccgcgtcctcga tggccaactt gccgtccgcc acccgttcaa 1320 ggtctttctg ataaaccttcacggacgcct cacccttgcc ccaatagaag gtccggcccg 1380 tgtcgctctc gatgacacgcggggccgcca tcttggacgc cttggacatg cggcgggcat 1440 agtccagcaa ggcgtccatcaaaccctctt ggctatggtc catcgacacg tcagcacgag 1500 ccagcaggga gggaccaaaggcgttcaggg cctgcggggc cagcagcgca caacgaccat 1560 caccaccggg tatttcaaggctcggcatgt ttctggcatg ccctgcccgc accgtagccc 1620 ggcgttcctc atcaatgggtgagtccgcgt aatgcaaggc acccgcgaaa ccgtccgtcc 1680 cgttgcccac gcgttgagtgtgcagaccag ccagcacaga ccagagacaa aaggcatccc 1740 gtgcctctct tgcctcttcctcgcccatcc tgtcctcgta gtgaatgaca cgcatggcct 1800 caacgcgttc agtgcccgttaagcgggcag cctcggcaac ctccgacggg tcagccctgc 1860 ggcgttcgcc cttgccgttccgcccgttag gcaacgtgat tgtgagccag tcgaaaaacc 1920 acgccgtttg cagccatttttccatgaccg ggctggtttt cttgcccttc cttcgcttct 1980 ccgaaggtgg ggggcactttggggcacaaa ccgccggttt tacgccgttc ggggcctgct 2040 tttcacattg aaaatcaggtgtttgccccg aactaccccc cctgttagca tgtggggggt 2100 tttgggcctt gcggcggacctcatcgagag ccgcgaccgc ctgctgtgaa agggttttac 2160 cgccgtggac catcatcgcgtctcccgttg agggtggacg cggtctcaag catgttgatt 2220 cttgcgaatg acatggctatatctctccaa agttctgagg acgacgcggg gaccgcgaaa 2280 tcacagcgtt cgtccaggttaagggctccg gttaatcccg gagcctttcc atttcatatg 2340 aacgacatcc ggaaaaatcaacatattgat gccgctggac gatgtgcggc caagatgtgc 2400 tgcagtattc ccggcgggcggaaccgttcc gatatttgaa gtctccacgc ttccgcccgc 2460 ctaatgtcgt tctgtcgttgaactttcata aaggtgaaac cctgctggtt tcccccggcg 2520 gcgaagccgc ctcccccttcctgcctcaca aaaaaaggga aaaggcccgt ttcaaccgat 2580 cgacgcaatc tcctttcacggtatccagat accttttctg gtgaagccat ccgaaacaaa 2640 ggccgcgagt cgtcacacctgggcttgacc gacgcgaatc actcggacag tgtccggtca 2700 tggaccacat gctgaccccaaaacaggcgg cggcccgtgc gggctgcggg cgttcctcta 2760 tcatgagggc gttaaagtcccaatctttgc ccgctatacg cgataatgag aaccgctggc 2820 agattgaccc ggacgcccttgaccgttggg ccggtcacag gccggacaat gaccggtcta 2880 tgaccgaaca cggaccggccacaccttcgg acacccaaac ggacaccccg gagaccttgg 2940 cacggctcgc cgttgccgaggcccggttga gtgatgccct gtcccgcgtc gaagatttgc 3000 agagagaacg ggatgaatggcgggcacagg cacaagcctt gacccgtcag cccggttggc 3060 tggaccgcct actgggtcgaacctgacccc ccatcgtcac ctttggcctc accagcggat 3120 tgcttgtgaa ccaaggccagcagttccttg acctgttcca cgctacccgc ttcggcttca 3180 atatcgccca tcttgacgcggacctttcgg ccttgtctcg cagccaccca tccaccgata 3240 gcaccaatca cagccggaccgagggttgtg gccatcatcg tgaactcacc caaaaaagtg 3300 atgccaccag agccagccgagtccctgatg aacgctcgct ccgaatattt tatgtcctgt 3360 tctttcagga tggtgcggaactccccggca tcttctttcg ggaaaaccgc gatttctaaa 3420 tcactcatgg aatgctccttttgcccatga ggtaataatg ccctgccaat gagcaccgag 3480 caaatcagcc gggttgctctttcgaaacgg tctcgggctt tggaagtgtc cacccctcga 3540 acagtgcccg gtctctctctggcatcgagg caatcgccat ccgcagcacc ttgacccatt 3600 gaggatcttt tgcagcttgcccgataaccg caccgcccag gacgattttc cgccgtgcgt 3660 ccttttttcg gtcctctgcccgaagttttg ccccggcttg tcgtatctgg gcctctgccc 3720 tcgccttggc ctctttggcccgctctaact gcctttctac tgaagttctt gccattttcc 3780 gtcctccttc atagggcctatatgaacgga gagggaacga agcaagaagt tctcaagaac 3840 ccgaagggcg cacttacacaaactctgggg agtttgtttc gggcgcccta ccgggggtca 3900 tctcgccgat ggggcgtctccgacggtaaa aagaggaggg gccgccgtgg cgatatatca 3960 cctgtccgcg tccattatcgggcgaagcga tggccggtca gccgttgccg cgtcagcata 4020 tcgggccggg gccgacatgaccgacccgga caccgggacg cggcacgact acacccgaaa 4080 gcgaggcgtc cgggcaaccttcatggagtt gcccgaaggt gcccccgatt gggccaccga 4140 ccgcccgagc ctctggaacgccgtccacgc gaaggagacg cggaagaact cgcggctttc 4200 gcgtgagatc c 4211 51458 DNA Ketogulonigenium misc_feature replicon pADMX6L1 5 abgggccttgcatccgatgg caagcaaaaa ctacccagtc cgtccgtagg cggggggtcg 60 ccagccctgtgggtgggcgc ttccccccgg cccgcaagcg ggcccggaat gggcattttt 120 tgcctgccctaagatcataa gaagggcaaa aaaaacatcg tttcaaaaca gcgtgttacc 180 acccccctataggacaccag agtccggggt agaggactct ggtgtcctct taggccattt 240 atgtccaagaatgtgacagc cagccgagcg gaggtagagg actctggtgt cctatgctta 300 ggccatttatgtccaaaaac ttgacaaggg ccacattcct gccaaatctg tccagaattt 360 ggaaaaattcgccggatagt agacagtggc aaagcctccc cccattcccg caaagcgccc 420 gctcggcacttgggttcaaa ctgaccggga agcccacgag gcgtgggcga tactggcaaa 480 aaagcctgctgccagcgctg tgatgcacat tctgtgcgcc aacctcggtg agcataatgc 540 cgtggtcatcagccaggaca ccatcgccaa gctgtgcggc ctttccacac ggtccgtcag 600 gcgcgccatcgtcgatctgg ccgaaggccg atggatcgag gttcgccaac ttggcgcgac 660 cagccagaccaatgcctatg tcgtcaacga ccgggtggca tggcagggat cacgggacgg 720 actgcgctacagcctgttta gtgcggctat agtcgtgtcc gaggaggagc aacccgaccg 780 cgctgaactcgaccagcaag cccccctgcg acacctgcca cgcatcagcg aggggcagat 840 acccaccggccccggcctgc cgccaccttc gcaaccgttt ctaaaagaca tggagccaga 900 cttgcccaccattgaccggg caacatcacc caactttgac cagcaggaac aggggtgaaa 960 aaggtggacaaactttccat acgcgaggcg gtaaaacact tcgatgtttc ccggccaacc 1020 ctgcaaaaagcccttaaatc tggcaagatt tcaggtgttc aggatggaca aggaacgtgg 1080 acaatagacccctcagagat ggcaagagtt taccagccaa ggcaagatga ggtggtaaag 1140 gatggtggccaagaacatga aaatttgtcc gccaagaaca cccctttaca tggtcaagtt 1200 gaggttctgaaagagcggct tgcagatgct gaaaaacggg tggcgatagc cgaggcactg 1260 gccgaagaacgtggaaaaca catcgaggat ctacgccgga tgctgcctgc accggaagcc 1320 ggtcagccccgccgccgctg gtggccatgg taaggtcagc tatgcgggac caagccgcag 1380 ctcgcaagtgcggcaacaga atcagacccg cttcggacag agagctcaag ctggtggaaa 1440 accgcctgtgaagctgct 1458 6 2401 DNA Ketogulonigenium 6 caccatggcg cgctgcagctcttctttgct tctgttggtt cttctctcgt caagaagctc 60 aggcggaaac cgcactataaagtgcagaac aggcttcttc gccgctttgt tttgcctaac 120 gccttttgtg tgcgcgtcatatgccgaccg aaggtctagc gtcttataaa cgagcggaga 180 ggcatctctt acgacgcgtttcgcacttgt tttgtcttgt cgtttcgcgt gcttttcggc 240 tgctgacaat ccagccatatctaacgcact acatctcacc gctgctttca ttttcaatcc 300 ctcatatatt accttttctgttgtttttgc gaaaaacgca aaactcgctt tgcgtttttt 360 gttcggcacc tgcggcacctccaaaaaaca cgcttgctct ccgagggtct ggcaggagcc 420 taagaggggg cattctgcccctgatcgacc ccattgaggc tcgatctaaa cagacccccc 480 acaggggcgt ctgtgggccgctagtgcggc cttccgccat cttcgagcca tctgatctct 540 gcaattagag atgcatggcgcatctcccat tccagctcag atatcccgta tctgtcgatg 600 acggtgtcat acaattcgtcgagccttgct tcgcgctctt cttttgtcat gccagttgca 660 tcgcgttctt ccatctcctcgttctcctct atgatgaaat cattttcgtc ctcgtcgttt 720 ggcccgcttt ctgctgccttgaccgcaaca gcgcctccgc gctctgcggc ctcgagcaag 780 ttcacgcgtt tagcgcgcacttgcttccac ccttcattgt cccactccgc gacaatttca 840 gggccgcttt gctctgctgcatcaacttca gccatagctt catcgtcatc cagctcgacc 900 aaaccgcatt cttctttcaagccttggctc cacaccaatt gccgcctacg cttgccgctc 960 gttgcattga aatattcgagccaaagcccg tcatcgcccg cctgaagtag ctgccttggc 1020 gtgcgtcctt tgcgttttccgctcttcgag cttgaaagcg tcaactcttc ggcagcgccc 1080 cacttcgcta cgtagtcgcccgcattggca gccccgcgaa cgtcaaacgc cgcatcgttg 1140 ccccacatgc cataccccttcagacatgca cgccacgcat cgcctagacg ttgcatcaga 1200 tgcagcgctt cgctttcatcgccagctctt agcaagacaa tttcgtgaaa gtgcgggtgc 1260 cacccatttg catagctatgagtaatttca gttgatgtga ctgacccaac aaatggtaaa 1320 tcgcgccact cgcggcgctgacgcaaccgc tgtttcgcct tcttcatgtt ttggagaaga 1380 tcaaaaagcg aatcacctgctttgtgctgg gctgtcagag ttatgagcac cggcacaaac 1440 ccgttgtcgc gcgcccacgcgagcaagtga ttcatttcag aacggcgaat ttgcgcgatg 1500 cgagcgctac aaactgcgcagccccacaca ttccggcact gtgctagacc tgaaaagaat 1560 gcccgacgcc cgccatcctcgcccacgttt tgcacgttta gctcaactgt cggagacact 1620 tttacgtgcc gacatttcgcaacttggtgg ggcttgtttt tgttcagatt caacagaatc 1680 cgagcggctg aacgcagatctgcataaagc tgccgcctac tgaataacct gttgttttct 1740 ttgttttttt cattcggttgacccccattc tggtcaaccg atttacggta tataccaagg 1800 ggggtctgcc gaccccctgaaaaagcgtca tcgccgcgcg cctgcgcacc cgcgttcttc 1860 ttcgatttct gaactgaatgtgatgctagt ttgtgagaca tggccgcaaa ccccgacggg 1920 tgcggcgact attctcttgattttctgcgt ctgtgcgcat actatcctcc gtgttcatca 1980 ggctcacgcc tgatctgattagggctcttg tctctgcttg tatcgtcgcc aaactatact 2040 ttaagcagcg tctagagcctgatgaacgat ctaagaagcc cgccccctga aaggcgggct 2100 tttttgatct gtgccagatgttgttacatc ggcgctcaaa aatcaagttt tttcttgact 2160 ttcaataatt tgcattatgcacattattat cgtttgataa taagagccag aacagcacag 2220 aatagttgtg cgatagctatgaataatagc agatccatcc ctgtttcctt tcttactaaa 2280 tacaatgcga aactgctcgcatctgtttta tttagttgaa tcggaaactc caaatcggcc 2340 ggattcaaaa aaaatatagactatctttaa agtagcaacg ccgccgctcg cgcgacggca 2400 t 2401 7 2029 DNAKetogulonigenium misc_feature replicon pADMX6L3 7 agagaataag atcaggtctgttctcgtgcc gccagcggtt aagcaggtta tagagccgaa 60 attcactctt attcatcaattgcttgctgc tgtagagcga ttttcgccct tcaacgaacc 120 agtagacacc cccgacaagggcgatgacca ccagaaatcc aattatcatt gtgaaattaa 180 tgtcggtcat gactgcactccctcaaggcc gaagtcagag attgcatctt gacgcagagc 240 gacgagttca acgccgtcgatgcggttttg ggtggcggta gccgggctgg cgcggatacg 300 cctaccgttg gcacggtttctaggcgacat actgaggcaa accggatttc cagtgggctg 360 cgtgctttgt tgtaagcgcgccatttcatc cccccgaggg caggggaacg gctcacaact 420 tccaaaggcc ccgaaaaattctcgcgatga ccccaccacg ggatttgtgc aacacaatcg 480 gcgcgcggga caggctcatatcaacttgga taagcgccgc gttttttgca aaatgcgaga 540 aagtgcttat ccgcatctttcacgttgatg gccttgtcat gcacccaaga acgccactct 600 tcttcaaggg agtaaacatcccacccagga gcaagttcac gggcagtatc gcgtgtgtcg 660 ggatcgttga acggcaaggcctgaaaagtg gttgatgcta tagtttccac caccttgggt 720 cggaagacag cgttctctccttcgatactc atgctgtagt cagggaagtg gtcgtgcgcg 780 gtgtcatcct caatgatcttcgaaagcagg cgacggaact cttttttagt tgagccggaa 840 ccgcacttgt ttcgcaagagctccaagcta cacatccatt ttgactgcgc gccacaatgc 900 tttcgcccta tctcatacaaccgcctttcg aggggctttc tgagcaggaa atacccccgg 960 cttagagtga ggacatggttgttctcgatc gcatcaaaga cccagtcaga gagcgttatt 1020 tcgacatcaa gcattcggccgtcgcgggtt acgcgcacga tctcggctga ttcaatcagg 1080 ccaaatacct tgaagtattctttcccacct tgacgaatat tcgtttcaat ttgggttccc 1140 tgcagccgcc gaagggcatctttgagcagc tgataaccct gaccggatgt ctgacggttc 1200 gttgccacaa gcagatcataagccttgaaa cgcatcgatc tacttatttt ctgcccctca 1260 ttaatggccg ccatgcactggctgatgcag tagatcagca catcacggtc atgaacggta 1320 gcaaggccat agcgtgaaggggaaacctcg atccaattgt cgttgttctg atagcggcgt 1380 ggcttcatgt ccggctttgtagacagggtg aacatagggt gctccatcga agccatatcc 1440 cccttgggaa ccgcatcaacgatgtcgcaa acgaaaaggt ctttttgtgg atgacgatcc 1500 ggcaaaagcg gtgatcgtaggttcgtcatt tcacacactc ccgcgccaag taaaaattcg 1560 tcatttcaca caccgtacaagactatcgtc atttcacaca ccactcgtca aggctcgtca 1620 tttcacacac ccaaggaggctgtggataac tcgagccgct atcgtcattt cacacaccat 1680 ctttcgtcat ttcacacaccatctttcgtc atttcacaca ccaggtgtta ttttttttat 1740 agttatatca attgattacgagctgttttc ggagctgtaa ctctattcta actctattct 1800 aactctcata gcttgccaaaatggcacctc atatcccgga tatccggttt cattatgaaa 1860 ccaatcaaca acatttacggtgttttttga ggagcaacac tgtcccaacg caggttcaaa 1920 ccgatcacgc cgaatctgcaaagaaagggg cagtgctatg ttcatttggt cactcgagga 1980 tcacccgaac cacaggtaagccctcacatg ctatgttgat acctccagg 2029 8 151 PRT Ketogulonigenium 8 MetHis Ile Leu Cys Ala Asn Leu Gly Glu His Asn Ala Val Val Ile 1 5 10 15Ser Gln Asp Thr Ile Ala Lys Leu Cys Gly Leu Ser Thr Arg Ser Val 20 25 30Arg Arg Ala Ile Val Asp Leu Ala Glu Gly Arg Trp Ile Glu Val Arg 35 40 45Gln Leu Gly Ala Thr Ser Gln Thr Asn Ala Tyr Val Val Asn Asp Arg 50 55 60Val Ala Trp Gln Gly Ser Arg Asp Gly Leu Arg Tyr Ser Leu Phe Ser 65 70 7580 Ala Ala Ile Val Val Ser Glu Glu Glu Gln Pro Asp Arg Ala Glu Leu 85 9095 Asp Gln Gln Ala Pro Leu Arg His Leu Pro Arg Ile Ser Glu Gly Gln 100105 110 Ile Pro Thr Gly Pro Gly Leu Pro Pro Pro Ser Gln Pro Phe Leu Lys115 120 125 Asp Met Glu Pro Asp Leu Pro Thr Ile Asp Arg Ala Thr Ser ProAsn 130 135 140 Phe Asp Gln Gln Glu Gln Gly 145 150 9 466 PRTKetogulonigenium 9 Met Ser His Lys Leu Ala Ser His Ser Val Gln Lys SerLys Lys Asn 1 5 10 15 Ala Gly Ala Gln Ala Arg Gly Asp Asp Ala Phe SerGly Gly Arg Gln 20 25 30 Thr Pro Leu Gly Ile Tyr Arg Lys Ser Val Asp GlnAsn Gly Gly Gln 35 40 45 Pro Asn Glu Lys Asn Lys Glu Asn Asn Arg Leu PheSer Arg Arg Gln 50 55 60 Leu Tyr Ala Asp Leu Arg Ser Ala Ala Arg Ile LeuLeu Asn Leu Asn 65 70 75 80 Lys Asn Lys Pro His Gln Val Ala Lys Cys ArgHis Val Lys Val Ser 85 90 95 Pro Thr Val Glu Leu Asn Val Gln Asn Val GlyGlu Asp Gly Gly Arg 100 105 110 Arg Ala Phe Phe Ser Gly Leu Ala Gln CysArg Asn Val Trp Gly Cys 115 120 125 Ala Val Cys Ser Ala Arg Ile Ala GlnIle Arg Arg Ser Glu Met Asn 130 135 140 His Leu Leu Ala Trp Ala Arg AspAsn Gly Phe Val Pro Val Leu Ile 145 150 155 160 Thr Leu Thr Ala Gln HisLys Ala Gly Asp Ser Leu Phe Asp Leu Leu 165 170 175 Gln Asn Met Lys LysAla Lys Gln Arg Leu Arg Gln Arg Arg Glu Trp 180 185 190 Arg Asp Leu ProPhe Val Gly Ser Val Thr Ser Thr Glu Ile Thr His 195 200 205 Ser Tyr AlaAsn Gly Trp His Pro His Phe His Glu Ile Val Leu Leu 210 215 220 Arg AlaGly Asp Glu Ser Glu Ala Leu His Leu Met Gln Arg Leu Gly 225 230 235 240Asp Ala Trp Arg Ala Cys Leu Lys Gly Tyr Gly Met Trp Gly Asn Asp 245 250255 Ala Ala Phe Asp Val Arg Gly Ala Ala Asn Ala Gly Asp Tyr Val Ala 260265 270 Lys Trp Gly Ala Ala Glu Glu Leu Thr Leu Ser Ser Ser Lys Ser Gly275 280 285 Lys Arg Lys Gly Arg Thr Pro Arg Gln Leu Leu Gln Ala Gly AspAsp 290 295 300 Gly Leu Trp Leu Glu Tyr Phe Asn Ala Thr Ser Gly Lys ArgArg Arg 305 310 315 320 Gln Leu Val Trp Ser Gln Gly Leu Lys Glu Glu CysGly Leu Val Glu 325 330 335 Leu Asp Asp Asp Glu Ala Met Ala Glu Val AspAla Ala Glu Gln Ser 340 345 350 Gly Pro Glu Ile Val Ala Glu Trp Asp AsnGlu Gly Trp Lys Gln Val 355 360 365 Arg Ala Lys Arg Val Asn Leu Leu GluAla Ala Glu Arg Gly Gly Ala 370 375 380 Val Ala Val Lys Ala Ala Glu SerGly Pro Asn Asp Glu Asp Glu Asn 385 390 395 400 Asp Phe Ile Ile Glu GluAsn Glu Glu Met Glu Glu Arg Asp Ala Thr 405 410 415 Gly Met Thr Lys GluGlu Arg Glu Ala Arg Leu Asp Glu Leu Tyr Asp 420 425 430 Thr Val Ile AspArg Tyr Gly Ile Ser Glu Leu Glu Trp Glu Met Arg 435 440 445 His Ala SerLeu Ile Ala Glu Ile Arg Trp Leu Glu Asp Gly Gly Arg 450 455 460 Pro His465 10 342 PRT Ketogulonigenium 10 Met Thr Asn Leu Arg Ser Pro Leu LeuPro Asp Arg His Pro Gln Lys 1 5 10 15 Asp Leu Phe Val Cys Asp Ile ValAsp Ala Val Pro Lys Gly Asp Met 20 25 30 Ala Ser Met Glu His Pro Met PheThr Leu Ser Thr Lys Pro Asp Met 35 40 45 Lys Pro Arg Arg Tyr Gln Asn AsnAsp Asn Trp Ile Glu Val Ser Pro 50 55 60 Ser Arg Tyr Gly Leu Ala Thr ValHis Asp Arg Asp Val Leu Ile Tyr 65 70 75 80 Cys Ile Ser Gln Cys Met AlaAla Ile Asn Glu Gly Gln Lys Ile Ser 85 90 95 Arg Ser Met Arg Phe Lys AlaTyr Asp Leu Leu Val Ala Thr Asn Arg 100 105 110 Gln Thr Ser Gly Gln GlyTyr Gln Leu Leu Lys Asp Ala Leu Arg Arg 115 120 125 Leu Gln Gly Thr GlnIle Glu Thr Asn Ile Arg Gln Gly Gly Lys Glu 130 135 140 Tyr Phe Lys ValPhe Gly Leu Ile Glu Ser Ala Glu Ile Val Arg Val 145 150 155 160 Thr ArgAsp Gly Arg Met Leu Asp Val Glu Ile Thr Leu Ser Asp Trp 165 170 175 ValPhe Asp Ala Ile Glu Asn Asn His Val Leu Thr Leu Ser Arg Gly 180 185 190Tyr Phe Leu Leu Arg Lys Pro Leu Glu Arg Arg Leu Tyr Glu Ile Gly 195 200205 Arg Lys His Cys Gly Ala Gln Ser Lys Trp Met Cys Ser Leu Glu Leu 210215 220 Leu Arg Asn Lys Cys Gly Ser Gly Ser Thr Lys Lys Glu Phe Arg Arg225 230 235 240 Leu Leu Ser Lys Ile Ile Glu Asp Asp Thr Ala His Asp HisPhe Pro 245 250 255 Asp Tyr Ser Met Ser Ile Glu Gly Glu Asn Ala Val PheArg Pro Lys 260 265 270 Val Val Glu Thr Ile Ala Ser Thr Thr Phe Gln AlaLeu Pro Phe Asn 275 280 285 Asp Pro Asp Thr Arg Asp Thr Ala Arg Glu LeuAla Pro Gly Trp Asp 290 295 300 Val Tyr Ser Leu Glu Glu Glu Trp Arg SerTrp Val His Asp Lys Ala 305 310 315 320 Ile Asn Val Lys Asp Ala Asp LysHis Phe Leu Ala Phe Cys Lys Lys 325 330 335 Arg Gly Ala Tyr Pro Ser 340

What is claimed is:
 1. Ketogulonigenium comprising a transgenecomprising a DNA sequence from an endogenous Ketogulonigenium plasmid.2. The Ketogulonigenium of claim 1, wherein said endogenousKetogulonigenium plasmid is contained in Deposit Number NRRL B-21627. 3.A method for producing the Ketogulonigenium of claim 1, comprising: (a)transforming a Ketogulonigenium strain with a transgene comprising a DNAsequence from an endogenous Ketogulonigenium plasmid; and (b) obtaininga stably transformed strain of Ketogulonigenium.
 4. The method of claim3, wherein said transforming comprises conjugation.
 5. The method ofclaim 3, wherein said transforming comprises electroporation.
 6. Themethod of claim 3, wherein said DNA sequence is derived from anendogenous plasmid contained in Deposit Number NRRL B-21627.
 7. A methodfor conjugative transfer of a vector from E. coli to Ketogulonigeniumcomprising culturing said E. coli with said Ketogulonigenium under suchconditions wherein said E. coli transfers said vector to saidKetogulonigenium.
 8. The method of claim 7, wherein said vectorcomprises pDELIA8.
 9. A method for transforming Ketogulonigeniumcomprising the process of inserting a vector into said Ketogulonigenium,wherein said insertion comprises electroporation, wherein said vectorcomprises pMF1014-α.
 10. An isolated or purified nucleic acid moleculecomprising a polynucleotide having a nucleotide sequence at least 95%identical to a sequence selected from the group consisting of thenucleotide sequence in SEQ ID NO:1, the nucleotide sequence in SEQ IDNO:2, the nucleotide sequence in SEQ ID NO:3, the nucleotide sequence inSEQ ID NO:4, a nucleotide sequence of the Ketogulonigenium portion ofthe plasmid contained in NRRL Deposit No. B-30418, a nucleotide sequenceof the Ketogulonigenium portion of the plasmid contained in NRRL DepositNo. B-30419, a nucleotide sequence of the Ketogulonigenium portion ofthe plasmid contained in NRRL Deposit No. B-30435, a nucleotide sequencecomplementary to SEQ ID NO:1, a nucleotide sequence complementary to SEQID NO:2, a nucleotide sequence complementary to SEQ ID NO:3, anucleotide sequence complementary to SEQ ID NO:4, a nucleotide sequencecomplementary to the Ketogulonigenium portion of the plasmid containedin NRRL Deposit No. B-30418, a nucleotide sequence complementary to theKetogulonigenium portion of the plasmid contained in NRRL Deposit No.B-30419, and a nucleotide sequence complementary to the Ketogulonigeniumportion of the plasmid contained in NRRL Deposit No. B-30435.
 11. Anisolated nucleic acid molecule comprising a polynucleotide whichhybridizes under stringent hybridization conditions to a polynucleotidehaving a nucleotide sequence identical to said nucleotide sequence ofclaim 10, wherein said polynucleotide which hybridizes, does nothybridize under stringent hybridization conditions to a polynucleotidehaving a nucleotide sequence consisting of only A residues or of only Tresidues.
 12. A vector comprising the molecule of claim 10 and at leastone marker gene.
 13. A vector comprising: (a) the nucleic acid moleculeof claim 10; (b) a terminator of transcription; (c) a promoter; and (d)a discrete series of restriction endonuclease recognition sites, saidseries being between said promoter and said terminator.
 14. An isolatedor purified nucleic acid molecule comprising a polynucleotide having anucleotide sequence at least 95% identical to a nucleotide sequence of aKetogulonigenium plasmid replicon found on any one of the endogenousplasmids contained in Deposit No. NRRL B-21627.
 15. The isolated orpurified nucleic acid molecule of claim 14 comprising a polynucleotidehaving a nucleotide sequence at least 95% identical to a sequenceselected from the group consisting of the nucleotide sequence in SEQ IDNO:5, the nucleotide sequence in SEQ ID NO:6, the nucleotide sequence inSEQ ID NO:7, a nucleotide sequence complementary to SEQ ID NO:5, anucleotide sequence complementary to SEQ ID NO:6, and a nucleotidesequence complementary to SEQ ID NO:7.
 16. An isolated nucleic acidmolecule comprising a polynucleotide which hybridizes under stringenthybridization conditions to a polynucleotide having a nucleotidesequence identical to said nucleotide sequence of claim 14, wherein saidpolynucleotide which hybridizes, does not hybridize under stringenthybridization conditions to a polynucleotide having a nucleotidesequence consisting of only A residues or of only T residues.
 17. Anisolated or purified vector comprising the nucleic acid molecule ofclaim
 14. 18. The vector of claim 16, comprising a replicon functionalin E. Coli.
 19. The vector of claim 16, comprising a replicon functionalin an organism selected from the genera comprising Acetobacter,Corynebacterium, Rhodobacter, Paracoccus, Roseobacter, Pseudomonas,Pseudogluconobacter, Gluconobacter, Serratia, Mycobacterium,Streptomyces and Bacillus.
 20. A transformed cell of E. coli comprisingthe vector of claim
 17. 21. A transformed cell of the genusKetogulonigenium comprising the vector of claim
 17. 22. The vector ofclaim 17, wherein said Ketogulonigenium replicon is selected from thegroup comprising the nucleotide sequences of SEQ ID NO:5, SEQ ID NO:6,and SEQ ID NO:7.